Effect of fluid motion on colony formation in Microcystis aeruginosa

Lin LI*, Wei ZHU, Ting-ting WANG, Yong-gang LUO Feng-lan CHEN Xiao TAN

1. College of Environment, Hohai University, Nanjing 210098, P. R. China

2. School of Geography and Environment, Jiangxi Normal University, Nanchang 330022, P. R. China

3. National Engineering Research Center of Water Resources Efficient Utilization and Engineering Safety, Hohai University, Nanjing 210098, P. R. China

4. Zhenjiang Academy of Urban Planning and Design, Zhenjiang 212001, P. R. China

Effect of fluid motion on colony formation in Microcystis aeruginosa

Lin LI*1,2, Wei ZHU1,3, Ting-ting WANG4, Yong-gang LUO1, Feng-lan CHEN1, Xiao TAN1

1. College of Environment, Hohai University, Nanjing 210098, P. R. China

2. School of Geography and Environment, Jiangxi Normal University, Nanchang 330022, P. R. China

3. National Engineering Research Center of Water Resources Efficient Utilization and Engineering Safety, Hohai University, Nanjing 210098, P. R. China

4. Zhenjiang Academy of Urban Planning and Design, Zhenjiang 212001, P. R. China

Microcystis aeruginosa, generally occurring in large colonies under natural conditions, mainly exists as single cells in laboratory cultures. The mechanisms involved in colony formation in Microcystis aeruginosa and their roles in algal blooms remain unknown. In this study, based on previous research findings that fluid motion may stimulate the colony formation in green algae, culture experiments were conducted under axenic conditions in a circular water chamber where the flow rate, temperature, light, and nutrients were controlled. The number of cells of Microcystis aeruginosa, the number of cells per colony, and the colonial characteristics in various growth phases were observed and measured. The results indicated that the colony formation in Microcystis aeruginosa, which was not observed under stagnant conditions, was evident when there was fluid motion, with the number of cells per largest colony reaching 120 and the proportion of the number of cells in colonial form to the total number of cells and the mean number of cells per colony reaching their peak values at a flow rate of 35 cm/s. Based on the analysis of colony formation process, fluid motion stimulates the colony formation in Microcystis aeruginosa in the lag growth phase, while flushes and disaggregates the colonies in the exponential growth phase. The stimulation effect in the lag growth phase may be attributable to the involvement of fluid motion in a series of physiological processes, including the uptake of trace elements and the synthesis and secretion of polysaccharides. In addition, the experimental groups exhibiting typical colonial characteristics in the lag growth phase were found to have higher cell biomass in the later phase.

Microcystis aeruginosa; colony formation; fluid motion; flushing effect; viscous shear; extracellular polysaccharide

1 Introduction

Blue-green algal blooms caused by lake eutrophication have posed severe risks to the environment in China (Guo 2007). The blue-green algal blooms in Taihu Lake, Chaohu Lake, and Tianchi Lake are mainly attributable to the overwhelming proliferation and aggregation ofMicrocystis aeruginosa, which generally occurs in large colonies with high algal cell densities in eutrophic lakes (Reynolds et al. 1981; Welker et al. 2004). The colonial characteristics of Microcystis aeruginosa, however, disappear under stagnant conditions, where it is difficult for Microcystis aeruginosa to aggregate or float, even when the cell density is high (Reynolds 2007; Bolch and Blackburn 1996). Colony formation in Microcystis aeruginosa plays an important role in the formation of blue algal blooms.

Some studies have suggested that zooplankton may induce colony formation in Microcystis aeruginosa (Becker 2010). But the colony-inducing effects of different zooplanktons may not be identical. Burkert et al. (2001) observed colony formation in Microcystis aeruginosa when investigating the interaction between the blue algae and the grazer (Ochromonas sp.). Yang et al. (2006), when conducting a mixed culture experiment of Microcystis aeruginosa and zooplankton, found that colony formation in Microcystis aeruginosa was absent with the presence of Copepoda, cladocerans, and Brachionus calyciflorus, but present with the presence of Ochromonas sp. In examining the colony-inducing effects of flagellate Ochromonas sp. on Microcystis aeruginosa, Microcystis wesenbergii, and Microcystis flos-aquae, colony formation was only observed in Microcystis aeruginosa (Yang et al. 2009). This suggests that colony formation may be highly specific, probably attributable to the presence of an infochemical referred to as kairomone by Lampert et al. (1994). In addition, colony formation in Microcystis aeruginosa was also observed with the presence of heterotrophic bacteria (Shen et al. 2011) and Ca2+in the medium (Wang et al. 2011), which indicates that many factors may contribute to colony formation.

In addition to the predation by zooplankton, Microcystis aeruginosa in natural lakes is also subjected to the turbulence of fluid motion, which, if too intensive, may have a negative effect on algal growth (Zhang et al. 2002). Fluid motion may also be conducive to colony formation as well as algal buoyancy and aggregation. Hondzo et al. (1998), when studying the effect of various shear rates on the growth of Scenedesmus quadricuda by employing a Couette apparatus, observed colonies comprised of dead and living cells. Hondzo and Lyn (1999) observed the colony formation in Scenedesmus quadricuda under turbulent flow generated by oscillating grids. In a similar study, the formation of spherical colonies in Phaeocystis globosa was also observed (Schapira et al. 2006). The above studies suggest that fluid motion plays an important role in colony formation of algal cells.

Lake hydrodynamic processes are very complicated. The wind-driven circulation of the lake and the turbulence generated at the lake inlet may, to a certain extent, influence the colony formation in Microcystis aeruginosa. However, whether the colony formation in Microcystis aeruginosa is associated with lake hydrodynamics as well as the mechanisms involved remains unknown. In this study, the effect of fluid motion on colony formation in Microcystis aeruginosa was investigated using a circular water chamber made of acrylic glass, where flow rates of various water bodies were simulated under controlled temperature, light, and nutrients conditions.

2 Materials and methods

2.1 Algae

Microcystis aeruginosa(the MA905 strain), which was obtained from the Institute of Hydrobiology of the Chinese Academy of Sciences, China, was inoculated in a BG-11 medium (Rippka et al. 1979) at a light intensity of 40 μmol/(m2·s) and a temperature of 25℃. Samples from the medium taken in the late exponential growth phase were washed using NaHCO3with a concentration of 15 mg/L and then centrifuged before being used for subsequent tests.

2.2 Experimental apparatus and methods

Two impellers with an adjustable motor were placed inside an acrylic glass circular water chamber to control the flow rate (Fig. 1). The circular chamber had a working volume of 1 500 mL.

The experiment was conducted at flow rates of 0, 5, 15, 25, 35, and 55 cm/s according to the range of horizontal flow rates observed in Taihu Lake (Qin et al. 2000). The control group with the flow rate of 0 cm/s was shaken manually three times every day to avoid algal sedimentation or adhesion to the bottom.

The total nitrogen (TN) and total phosphorus (TP) concentrations were, respectively, adjusted to 7 mg/L and 0.5 mg/L in the BG-11 medium with NaNO3and K2HPO4. The algae were inoculated in the circular chamber inside an incubator, with an initial density of 2 × 105cells/mL, at a temperature of 25℃, a light intensity of 50 μmol/(m2·s), and a light-dark cycle of 14L:10D. Each experimental group was cultured at a pre-set flow rate except during the sampling and replenishment of the culture medium, when the fluid motion was stopped. Before the experiment, the apparatus was sterilized for two hours using a UV lamp, and the culture medium was sterilized with an autoclave. The experiment was conducted under axenic conditions. The tests were run in triplicate for each experimental group.

Two milliliters of sample from the medium was taken using an axenic pipet at 8:00 a.m. every day, and then transferred to a test tube, where Lugol’s reagent was added for preservation. The number of cells was measured by counting at least 300 units under an electron microscope. Colonies with three algal cells or more were counted to determine the number of colonies as well as the number of cells per colony. The proportion of the number of cells in colonial form to the total number of cells and the mean number of cells per colonywere calculated. All data were presented as mean ± standard deviation. Analysis of variance (ANOVA) was used to determine the significance of the differences in the biomass and mean number of cells per unit between the control and the treatment groups.

Fig. 1Acrylic glass circular water chamber (Unit: mm)

3 Results

One day after inoculation, Microcystis aeruginosa colonies were observed in each experimental group, as shown in Figs. 2(a) and 2(b). Colony formation in Microcystis aeruginosa of each experimental group was evident. In contrast, Microcystis aeruginosa was only present as single cells or paired cells (Fig. 2(c)) in the control group. As shown in Fig. 2(d), the colonies found in Taihu Lake are significantly larger than those cultured in the laboratory.

Fig. 2 Images of Microcystis aeruginosa in different experimental groups and Taihu Lake

Biomass, quantified in terms of the algal cell density, was used to indicate the growth of Microcystis aeruginosa during the experimental period (Fig. 3). The cell density of Microcystis aeruginosa varied with the flow rate of each experimental group. The biomass first increased and then declined with the increase of the flow rate. In this study, the optimum flow rate for colony formation in Microcystis aeruginosa was 35 cm/s.

In each experimental group, the number of cells present in the largest Microcystis aeruginosa colony, also referred to as the number of cells per largest colony, was counted to identify the characteristics of Microcystis aeruginosa colonies at different flow rates. Asshown in Fig. 4 (where bars indicate the standard error), the number of cells per largest colony exhibited a similar tendency to the algal cell density, which increased with the flow rate until a maximum of 120 algal cells was reached and then declined with the increase of the flow rate. In the control group, however, allMicrocystis aeruginosacells were single.

Fig. 3Variation of algal density with culture time at different flow rates

Fig. 4Number of cells per largest colony at different flow rates in early logarithmic growth phase

Fig. 5Variation of proportion of number of cells in colonial form (ρc) to total number of cells (ρt) at different flow rates

The colonial characteristics in terms of the number of cells per colony are illustrated in Fig. 6. After one day of culture, all experimental groups were dominated by colonies with three to nine algal cells, which accounted for over 0.8 of the total colonies formed except for the group at a flow rate of 35 cm/s, for which the proportion of colonies with three to nine cells accounted for about 0.5 During the culture period, the proportion of colonies with larger number of cells gradually increased, with colonies consisting of ten or more cells accountingfor over 0.2 (0.57 at the flow rate of 35 cm/s) on the 5th day. From the 5th day onward, the number of cells started to decline, and almost returned to its original state (on the 1st day) on the 14th day.

Fig. 6Proportion of three types of colonies at different flow rates

As shown in Fig. 7, the mean number of cells per colony increased gradually with the increase of the flow rate until a maximum value of 18 occurred at the flow rate of 35 cm/s on the 5th day and then declined till the end of the experimental period. The mean number of cells, which varied with culture time, reached their maximum values on the 9th day for the experimental groups with flow rates of 5 cm/s, 15 cm/s, and 25 cm/s. As the flow rate increased, the maximum mean number of cells for the experimental groups with flow rates of 35 cm/s and 55 cm/s occurred earlier, on the 5th day and the 3rd day, respectively. This suggests that the flow rate affects the number of cells in colonies, which vary significantly in different growth phases.

This experiment suggests that the flow rate had a siginficant effect on the formation ofMicrocystis aeruginosacolonies, which mostly presented as single or paired cells under stagnant conditions. This result is consistent with the test results of previous studies (Burkert et al. 2001; Yang et al. 2009). In this experiment, the colonies formed are dominated by those consisting of approximately three to nine cells, with the largestMicrocystis aeruginosacolony of up to 120 cells. Although significantly different from the structure of colonies in naturallakes (Reynolds and Jaworski 1978; Joung et al. 2006), the structure of the colonies in this experiment is similar to that mix cultured with zooplankton by Yang et al. (2005, 2006), except with higher numbers of cells per colony.

Fig. 7 Mean number of cells per colony at different flow rates

4 Discussion

In this experiment, while colony formation was observed under fluid motion conditions, it was not observed under the stagnant condition, which was probably because the fluid motion increased the chance of contact between algal cells, causing algal cells to form colonies. Although fluid motion faciliated colony formation in the lag growth phase, it disaggregated the colony in the exponential growth phase, when algal cells mainly occurred as paired or single cells. Two mechanisms may be involved in the formation of Microcystis aeruginosa colonies: (1) the alignment of cells, which remain regularly attached to the mother cell after cell division during the proliferation; and (2) the adhesion of existing single cells due to the extracellular slime substances (Lürling 2003). As shown in Figs. 2(a) and 2(b), formed randomly in Microcystis aeruginosa under the effect of fluid motion, the colony structure was significantly different from that in natural environment (Fig. 2(d)). This may be associated with extracellular polysaccharides, which are conducive to algal cells to maintaining typical colonial morphology (Ki?rboe et al. 1990; Jackson and Lochmann 1993; Hosaka et al. 1995). The colony formation in Microcystis aeruginosa from single cells may be induced by many factors, including the predation by zooplankton (Burkert et al. 2001), the presence of infochemicals (Yang et al. 2005, 2009), the bridging effect of Ca2+(Wang et al. 2011), and the polysaccharide substances secreted by attached heterotrophic bacteria (Shen et al. 2011).

Fluid motion may affect the morphology of Microcystis aeruginosa colonies in two ways (as shown in Fig. 8): on the one hand, it stimulates colony formation in Microcystis aeruginosa in the lag growth phase; on the other hand, it disaggregates the colony structure of Microcystis aeruginosa in the late exponential growth phase. The colony-inducing effect of fluid motionon Microcystis aeruginosa in the lag growth phase may be attributable to the enhanced secretion of polysaccharides, and a physiological adaptive response to various environmental factors. According to Ruiz et al. (2004), Microcystis aeruginosa can adapt to fluid motion by changing its physiological characteristics. Therefore, appropriate fluid shear may induce the secretion of extraceullar substances by Microcystis aeruginosa. Moore and Tischer (1964) observed, when studying Chlorophyta and blue-green algae, that the extracellular polysaccharide accumulation was enhanced due to the turbulence in the lag growth phase, which was similar to the results of Sangar and Dugan (1972) in studying Anacystis. Appropriate fluid motion is conducive to colony formation in Microcystis aeruginosa because it faciliates cell division, polysaccharide synthesis, cell contact, etc. Whether the induction of polysaccharide secretion by fluid motion is associated with the enhanced uptake of trace elements by Microcystis aeruginosa in the culture medium under tubulent conditions still needs to be investigated.

Fig. 8 Colony formation process of Microcystis aeruginosa in different growth phases

In the late exponential growth phase, when the intercellular cohesion decreased with the acceleration of the algal cell division and the decrease of the synthesis rate of exopolysaccharides, the fluid motion mainly exhibited a flushing effect (viscous shear) on the colonies, thus disaggregating the colonies formed. At the flow rate of 35 cm/s, where the induction of colony formation due to fluid motion was relatively strong and the flushing effect was relatively weak, the colonial characteristics of Microcystis aeruginosa were most typical. Besides the most typical colonial characteristics, the highest Microcystis aeruginosa biomass was observed at the flow rate of 35 cm/s. This indicates that Microcystis aeruginosa featuring more typical colonial characteristics at the early culture stage is mostly likely to achieve a higher algal cell density. Therefore, the colony formation in Microcystis aeruginosa in the lag growth phase, which is beneficial to its adaption to fluid motion, may accelerate the growth and proliferation of cells.

The relationship between cell biomass and colony structure was illustrated in Fig. 9, with the horizontal coordinate representing the mean number of cells per colony in the lag growth phase (colonies from the 1st day to the 5th day), the longitudinal coordinate representing thealgal cell density in each phase corresponding to the mean number of cells per colony, and R2representing the coefficient of determination. The significant level of correlation (p ＜ 0.01) was observed between the horizontal coordinate and the longitudinal coordinate. The colonies with a larger mean number of cells in the lag growth phase had higher algal densities in all growth phases. The colony formation in Microcystis aeruginosa may be one of the major mechanisms for mass proliferation of Microcystis aeruginosa.

Fig. 9 Relationship between mean number of cells per colony in lag growth phase and mean algal cell densities in exponential and stationary growth phases

5 Conclusions

(1) A culture experiment was conducted in a customized apparatus with controlled temperature, light, and flow rate. Colony formation in Microcystis aeruginosa was observed in the experimental groups at different flow rates in the apparatus, with the largest colony consisting of 120 algal cells. In the control group without fluid motion, Microcystis aeruginosa existed mostly as single or paired cells. The flow rate may have a significant effect on the colony formation in Microcystis aeruginosa.

(2) As fluid motion increased the chance of contact between algal cells, it facilitated the formation of colonies in the lag growth phase. In the late exponential growth phase, the fluid motion exhibited a flushing effect (viscous shear) on the colonies formed, which may occur earlier with the increase of flow rate.

(3) The experimental groups featuring typical colonial characteristics and more algal cells achieved higher cell biomass. This tendency appeared at various flow rates and in different growth phases.

Acknowledgements

We gratefully acknowledge Dr. Taisuke Ohtsuka from the Lake Biwa Museum for many suggestions and fruitful discussions.

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(Edited by Yun-li YU)

This work was supported by the National Natural Science Foundation of China (Grant No. 50979028) and the Special Fund of Research for Public Welfare Industry of the Ministry of Water Resources of China (Grant No. 200801065).

*Corresponding author (e-mail: Li_lin980192@126.com)

Received Oct. 8, 2011; accepted Feb. 28, 2012