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Environmental factors controlling colony formation in blooms of thecyanobacteria Microcystis spp. in Lake Taihu, China

Jianrong Ma a,b, Justin D. Brookes c, Boqiang Qin a,*, Hans W. Paerl d, Guang Gao a,Pan Wu a,b, Wei Zhang a, Jianming Deng a,b, Guangwei Zhu a, Yunling Zhang a,Hai Xu a, Hailin Niu a,b,e

a State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology,

Chinese Academy of Sciences, 73 East Beijing Road, Nanjing 210008, PR Chinab University of Chinese Academy of Sciences, Beijing 100049, PR Chinac School of Earth and Environmental Science, University of Adelaide, Adelaide 5005, Australiad Institute of Marine Sciences, The University of North Carolina at Chapel Hill, Morehead City, NC 28557, USAe College of Ecological and Environmental Engineering, Qinghai University, Xining 810016, PR China

1. Introduction

The cycles of the key macronutrients nitrogen (N) andphosphorus (P) have been massively altered by anthropogenicactivities (Elser et al., 2007). Over-enrichment by nitrogen andphosphorus accelerates eutrophication of water bodies andpromotes algal blooms, which has been observed in lakesworldwide (Conley et al., 2009a,b; Hautier et al., 2009; Paerlet al., 2011a; Schindler and Hecky, 2009). Cyanobacteria producetoxins and taste and odor compounds which threaten drinkingwater supply and adversely affect recreational and fishing use ofaffected systems. Large accumulations of cyanobacteria, surface

blooms, can lead to hypoxia and disrupt food webs. Cyanobacteriarepresent a public health threat and cause undesirable ecological,economic, water resource impacts and related societal problems(Brookes and Carey, 2011; Paerl et al., 2011a). In large eutrophiclakes, such as Lake Taihu, China where blooms are widespread andpersistent, there is an increased urgency to deal with this problem.

Many bloom-forming cyanobacterial genera exist as macro-scopic colonies that are present in buoyant surface blooms ordispersed throughout the water column. Microcystis is a commonand often dominant colonial, bloom-forming genus responsible forharmful (toxic, food-web disrupting, and hypoxia generating)blooms in nutrient-enriched lakes worldwide (Paerl et al., 2011b).The mechanisms underlying how and why Microcystis formcolonies remain poorly understood. Microcystis generally existsas single cells, or a few paired cells, in laboratory cultures butoccurs in colonies under natural conditions (Reynolds et al., 1981;

Harmful Algae 31 (2014) 136–142

A R T I C L E I N F O

Article history:

Received 24 March 2013

Received in revised form 23 October 2013

Accepted 23 October 2013

Keywords:

Blooms

Colonies

Eutrophication

Microcystis

Nitrogen

Phosphorus

A B S T R A C T

Nitrogen (N) and phosphorus (P) over-enrichment has accelerated eutrophication and promoted

cyanobacterial blooms worldwide. The colonial bloom-forming cyanobacterial genus Microcystis is

covered by sheaths which can protect cells from zooplankton grazing, viral or bacterial attack and other

potential negative environmental factors. This provides a competitive advantage over other

phytoplankton species. However, the mechanism of Microcystis colony formation is not clear. Here

we report the influence of N, P and pH on Microcystis growth and colony formation in field simulation

experiments in Lake Taihu (China). N addition to lake water maintained Microcystis colony size,

promoted growth of total phytoplankton, and increased Microcystis proportion as part of total

phytoplankton biomass. Increases in P did not promote growth but led to smaller colonies, and had no

significant impact on the proportion of Microcystis in the community. N and P addition together

promoted phytoplankton growth much more than only adding N. TN and TP concentrations lower than

about TN 7.75–13.95 mg L�1 and TP 0.41–0.74 mg L�1 mainly promoted the growth of large Microcystis

colonies, but higher concentrations than this promoted the formation of single cells. There was a strong

inverse relationship between pH and colony size in the N&P treatments suggesting CO2 limitation may

have induced colonies to become smaller. It appears that Microcystis colony formation is an adaptation to

provide the organisms adverse conditions such as nutrient deficiencies or CO2 limitation induced by

increased pH level associated with rapidly proliferating blooms.

� 2013 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +86 025 86882192; fax: +86 025 57714759.

E-mail address: [email protected] (B. Qin).

Contents lists available at ScienceDirect

Harmful Algae

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ha l

1568-9883/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.hal.2013.10.016


Wu et al., 2007). Colonies with sheaths and mucilage can preventcells from zooplankton grazing, viral or bacterial attack, desiccationand other potential negative environmental factors, and increasefloating velocity, all of which provide a competitive advantage overother phytoplankton genera (Cyr and Curtis, 1999; Kearns andHunter, 2001; Wu and Kong, 2009; Yamamoto et al., 2011). Variousabiotic and biotic factors are important in affecting changes inphytoplankton morphology; specifically determining colonial vs.single cell forms (Fiałkowska et al., 1997; Trainor, 1993; Yang et al.,2008). Some studies have suggested that Microcystis colonyformation may be stimulated by flagellate grazing (Burkert et al.,2001; Wang et al., 2010; Yang et al., 2006, 2008). The cyanotoxins,microcystins (MCs), enhance colony formation in Microcystis spp.and may play a key role in the persistence of algal colonies and thedominance of Microcystis (Gan et al., 2012).

Many studies have explored the impact of N and P onMicrocystis growth (Gerloff and Skoog, 1954; Vezie et al., 2002;Xu et al., 2010). However, there is little knowledge on the impact ofnutrient enrichment on Microcystis colony formation. Lake Taihu, alarge shallow, hypereutrophic lake in China, has suffered annualcyanobacterial blooms dominated by Microcystis aeruginosa,Microcystis flos-aquae and Microcystis wesenbergii, which togethercontribute more than 40%, and up to 98%, of the totalphytoplankton biovolume from May to October (Chen et al.,2003). In the present investigation, which used native Microcystis

spp. from Lake Taihu, we show that high concentrations of P and Nplus P as well associated high pH significantly decreased the size ofMicrocystis colonies. We examined potential mechanisms under-lying the relationship between N and P enrichments andMicrocystis colony formation.

2. Experimental procedures

2.1. Experimental design

A series of bioassays were set up with treatments of lake watercontaining varying N and P concentrations to assess how nutrientadditions impacted Microcystis colony size. Thirty liters (30 L) of lakewater containing Microcystis colonies was pumped into white plasticbuckets (48 buckets, each bucket had a maximum volume of 35 L) on10 September 2012 and incubated outside at the Taihu Laboratory ofLake Ecosystem Research (TLLER) on the shores of Lake Taihu, Wuxi.N and P were added as NaNO3 and KH2PO4. Three nutrient additiontreatments (N only, P only and both N&P) manipulated the nutrientconcentrations to 2, 4, 8, 16 and 32 times to the ambient lake water(Table 1). The control treatment contained lake water with nonutrient addition. Each treatment was triplicated. Physico-chemicalparameters were measured on days 0, 3, 6, 9, 12, 15, 18, between8:00 am and 9:00 am. Coinciding with the sampling of physico-chemical variables, 0.5 L of water was sampled from each bucket todetermine Microcystis colony size, cell concentration, phytoplanktoncommunity composition and chlorophyll a. Weather conditions andair temperature were recorded every day. Each bucket was stirreddaily at 7:00 and 19:00.

2.2. Measurements

Physico-chemical parameters, including water temperature(WT), dissolved oxygen (DO), pH and electrical conductivity (EC),were measured with a Yellow Springs Instruments (YSI) 6600multi-sensor sonde. 0.5 L water was sampled initially from eachbucket to determine biological and chemical parameters, includingMicrocystis colony size, phytoplankton community composition,and chlorophyll a.

The size of Microcystis colonies was determined using imageanalysis employing Olympus DP Soft software, with an OlympusBX 51 light microscope and an Olympus DP 71 digital camera, asdescribed by Wilson et al. (2010). To count Microcystis colonies,1 mL aliquots were counted by microscopy on days 0, 3, 6, 9, 12, 15and 18 using a wide-mouth pipette. Fifty colonies were randomlychosen from each sample and sized with a calibrated ocular (200�magnification). Colony size was measured as equivalent sphericaldiameter (ESD) of the colony [ESD = (6V/p)1/3].

Phytoplankton samples were preserved with Lugol’s iodinesolution (2% final conc.) and sedimented for 48 h. Cell density wasmeasured with a Sedgwick–Rafter counting chamber undermagnification of 200–400� Phytoplankton species were identifiedaccording to Hu et al. (1980). Algal biovolumes were calculated fromcell numbers and cell size measurements. Conversion to biomassassumed that 1 mm3 of volume was equivalent to 1 mg of freshweight biomass. The proportions of Microcystis to total phytoplank-ton were calculated by Microcystis biovolumes to total algalbiovolumes. Chl-a concentrations were determined spectrophoto-metrically after extraction in 90% hot ethanol (Papista et al., 2002).

2.3. Statistical analysis

Data are presented as means � SD. Significant differencesbetween control and treated samples were determined by ANOVAwith Tukey post hoc test. Statistical analyses were conducted withSPSS17.0.

3. Results

3.1. Environmental factors

The weather conditions were cloudy (day 0–2, day 4, day 11,day 17–18), rainy (day 3 and day 12) and sunny (day 5–10, day 13–16). Air temperature ranged from 16 to 28 8C. Water temperatureranged from 18.8 to 22.5 8C. The pH, dissolved oxygen (DO)concentration, dissolved oxygen saturation (DO%), and electricalconductivity (EC) in N plus P additions were greater than thoseobserved in either N additions, or P additions and controls(Table 2).

3.2. N, P and N&P impacts on Microcystis colony size

Microcystis aeruginosa, Microcystis flos-aquae and Microcystis

wesenbergii were the main Microcystis species in this study.

Table 1Overview of the experimental treatments and different form of N and P of lake water (unit: mg L�1).

TP TN TDP PO4-P TDN NH4-N NO3-N

Water from lake 0.082 1.55 0.053 0.008 0.95 0.87 0.063

2 times added 0.164 3.1 0.164 3.1

4 times added 0.328 6.2 0.328 6.2

8 times added 0.656 12.4 0.656 12.4

16 times added 1.312 24.8 1.312 24.8

32 times added 2.624 49.6 2.624 49.6

Nitrogen and Phosphorus were added as NaNO3 and KH2PO4 respectively corresponding to concentrations 2, 4, 8, 16 and 32 times the ambient concentration. NaNO3 and

KH2PO4 were dissolved in pure water before added to buckets to increase concentrations of N, P, N&P to the set level.

J. Ma et al. / Harmful Algae 31 (2014) 136–142 137


Microcystis colony sizes from samples of natural lake water rangedfrom 20 to 1100 mm with an average of 205 � 21 mm. Colony sizedecreased in every treatment including the control. P only and highconcentration of P plus N additions promoted Microcystis colony sizedecreased significantly (relative to control). There was no significant(p > 0.05) difference between N addition alone and the control(Fig. 1a). However, Microcystis colony size in only P treatments weresignificantly smaller than the control after 9 days (Fig. 1.b; day 9–12,p < 0.05 in 2–4� p < 0.01 in 8–32� and p < 0.01 after 15 days in allonly P addition). Increasing concentration (8–32�) of both N and Prelative to lake water (TN � 13.95 mg L�1, TP � 0.74 mg L�1 inbuckets) caused a decrease in colony size relative to control andMicrocystis existed in the form of single cell and extremely smallcolonies after 15 days. The 32� addition (TN = 51.15 mg L�1 and

TP = 2.706 mg L�1 in buckets) led to a decrease in colony size fromday 3 (p < 0.05) but 8–16� from day 9 (p < 0.01). However,Microcystis colony size in treatments with both N and P additionsat concentrations two to four times ambient concentrations were notsignificantly different from the control (Fig. 1c). The morphology ofthe colonies also changed over the course of the experiment (Fig. 2).Colony size in treatments 32� P, 32� N&P and control all showed areduction in size. The characteristic change in colony morphologywas from a clathrate form initially to smaller more loosely packedcolonies later in the period.

There were no significant correlations (p > 0.05) betweenenvironmental factors and Microcystis colony size in all treatmentswith the exception of pH values which was negatively correlatedwith colony (Fig. 3).

3.3. N, P and N&P impacts on phytoplankton biomass and dominance

of Microcystis

The initial phytoplankton biomass concentration (Chl-a) in lakewater was 20.46 � 0.82 mg L�1. The addition of N alone (2–32�ambient) resulted in growth that exceeded the control after 12 days ofincubation (Fig. 4a; p < 0.05). The biomass of treatments in whichonly P was added did not differ significantly from controls (Fig. 4b;p > 0.05). Biomass in both N&P added treatments was much higherthan control and N only additions significantly from day 3 (p < 0.05).The treatment with 8 times N&P added (TN = 13.95 mg L�1,TP = 0.74 mg L�1 in buckets) had the highest Chl-a concentrationon day 15 (281.6 � 28.9 mg L�1; Fig. 4c).

Total phytoplankton biovolume was 36.6 � 3.9 mg L�1 and theMicrocystis proportion was 30.89 � 1.5% initially. The 2� N additionhad no significant (p > 0.05) effect on the proportion of Microcystis

but 4-32 times N additions was higher than control on day 12 (Fig. 5a;p < 0.05). There was no significant difference between the P additiontreatment and the control (Fig. 5b; p < 0.05). The proportion ofMicrocystis declined until day 6 in the N&P addition treatment butthen steadily increased (Fig. 5c).

4. Discussion

Interestingly, the additions of P and high concentration of P plusN resulted in Microcystis colonies smaller than the control, which isin contrast with the N only treatments and treatments with lowconcentration of P plus N (Fig. 1). Larger colonies persisted intreatments with N addition alone and low concentrations of N + P.In the high concentration N + P treatments the colonial form wasalmost entirely absent. The observed change in colony size couldbe a combined effect of both aggregation and disaggregation ofexisting colonies, and the production of new colonies. Such asfilaments of Anabaena circinalis aggregated under low lightconditions, effectively increasing their size and buoyancy (Brookeset al., 1999).

Colony formation is a trade-off between nutrient uptake,diffusion of gases into and throughout the colony, light require-ments, predator avoidance, buoyancy regulation and the ability tovertically migrate rapidly enough to exploit resource (e.g.,nutrient) availability in the water column, which are often

Table 2Range of pH, DO, DO% and EC in various treatment.

Parameters Control N additions P additions N plus P additions

pH 8.91–9.41 8.79–9.46 8.97–9.54 9.45–11.28

DO (mg L�1) 8.95–12.24 9.27–14.66 9.08–11.13 9.1–19.64

DO% (%) 102.9–113.4 105.3–120.9 101.1–124.7 101.4–232.3

EC (mS cm�1) 435–638 439–1211 428–698 488–1346

The pH, dissolved oxygen (DO), dissolved oxygen saturation (DO%), and electrical conductivity (EC) were measured between 8:00 am and 9:00 am on days 0, 3, 6, 9, 12, 15, 18

by a Yellow Springs Instruments (YSI) 6600 multi-sensor sonde.

Fig. 1. Colony size variations of Microcystis in buckets added only N (a), only P (b)

and N&P (c). Added amount were 2, 4, 8, 16 and 32 times relative to N, P or N&P of

lake water in Taihu, China. Controls are same in A–C.

J. Ma et al. / Harmful Algae 31 (2014) 136–142138


vertically separated (Ganf and Oliver, 1982). Resource availabilityimpacts upon these aspects of growth, polysaccharide accumula-tion and colony formation. N and P are essential elements for thesynthesis of nucleic acids, ATP or proteins, which are necessary forcell division and growth (Conley et al., 2009b). Extracellularpolysaccharides (EPS), comprised of C, H and O, are the mainconstituents of the colonial Microcystis sheaths associated with cellaggregates (Plude et al., 1991). Both soluble carbohydrates andtotal carbohydrates have been shown to be significantly higher incells and sheaths of colonial Microcystis than in disaggregated cells(Zhang et al., 2007). When there is an adequate supply of N and P(more than 7.75–13.95 TN mg L�1 and 0.41–0.74 TP mg L�1 in thisstudy) for cell division and growth, the total phytoplanktonbiomass is able to increase until a resource, such as light, becomeslimiting (Van de Waal et al., 2009). Under these nutrient repleteconditions photosynthetic products are transformed to proteins,

nucleic acids and ATP for cell proliferation and growth and normalactivities. Excess accumulation of EPS is less in these environmentsand there is a reduced need for Microcystis to form colonies whichlimiting growth (Li et al., 2013). In contrast, Microcystis mayproduce more EPS when nutrient supply is inadequate. N or Pstarvation has often been described as a condition that enhancesEPS synthesis (De Philippis et al., 2001; Huang et al., 2007; Oteroand Vincenzini, 2003), and has been shown to lead to carbohydrateaccumulation within cells (Brookes and Ganf, 2001). Low Pavailability led to an increase in EPS production in many cases(De Philippis et al., 2001; Huang et al., 2007; Nicolaus et al., 1999;Roux, 1996). However, the relationship between phosphorusavailability and the production of EPS is not straightforward (Grilloand Gibson, 1979), and there are examples where an increase in Pconcentration had little effect on the amount of exopolymers(Pereira et al., 2009). While a mechanism leading to a reduction in

Fig. 2. The morphology of colonial Microcystis in buckets added 32 times P, 32 times N&P treatment and control in day 0, 3, 9 and 18. All colonies were common and chosen

randomly, bar = 20 mm.



colony size has not been identified in the present study, it isplausible a change in colonial EPS is implicated. N availability mayalso affect EPS production and several authors have observed anincrease in EPS synthesis with increasing N concentrations (Oteroand Vincenzini, 2003; Suresh Kumar et al., 2007). The amount ofpolymer produced varied according to the N source supplied(Philippis and Vincenzini, 1998). The bioassays revealed Nlimitation (Fig. 4a), and N limitation has also been observed inLake Taihu during Microcystis bloom (Xu et al., 2010). Interestingalthough growth rate varied between the control treatments andthe N addition treatments, there was no difference in colony size.

However, colony size decreased steadily with time in alltreatments, including in the control treatment. This may have beendue to CO2 limitation, even in the controls, because there wasgrowth on the controls and pH went up (Talling, 1976). The pH alsonegatively correlated with the size of the colonies, especially in Nplus P additions (Fig. 3). Carbon limitation may limit photosyn-thetic production of extracellular polysaccharides to form colonialMicrocystis sheaths. The preference for CO2 over HCO3

� and CO32�

as a photosynthetic carbon source but there is little free CO2 inwater when pH exceeds 10 (Paerl and Ustach, 1982). In addition, itis extremely difficult for cells inside of large Microcystis colonies to

Fig. 3. The relationship between pH value and colonies size of virous treatment

bucket. There is no significant (p > 0.05) relationships between pH and colonies size

in control (n = 21), P additions (n = 105) and N additions (n = 105). In contrary, clear

linear relationship in N plus P additions (n = 105).

Fig. 4. Chl-a concentration dynamic in buckets added N (a), P (b) and N&P (c).

Controls are same in A–C.

Fig. 5. Microcystis proportion (bio volume) dynamic in buckets added N (a), P (b) and

N&P (c). Controls are same in A–C.



obtain CO2 under high pH condition (Gavis and Ferguson, 1975).Thus, at high pH levels which were promoted by N&P additions,there would be an advantage to existing as individual cells asopposed to colonies in order to minimize constraints on CO2

diffusion.Grazing also plays a role in colony formation (Fulton and Paerl,

1987; Yang et al., 2008). Watson (Watson et al., 1992) suggestedthat below concentrations of 8–10 mg L�1 TP, total biomass isdominated by edible algae (<35–50 mm). Above this range, there isa transition zone where both edible and inedible (>35–50 mm)algae are similar in relative abundance. With increasing concen-trations to approximately 30 mg L�1, the inedible algae rapidlybecome more dominant, until at TP > 50 mg L�1 the phytoplanktonbiomass consists almost entirely of this larger size fraction. In ourexperiments, colony size of Microcystis in 2 times N&P additionalways and 4 times (TN = 7.75 mg L�1 and TP = 0.41 mg L�1 inbuckets) addition were slightly higher than the control in previous9 days (Fig. 1c). The nutritional value of colonies may vary withsize, with large colonies being able to store more nutrients, therebypreventing predation and countering other potential negativeenvironmental factors effectively (Cyr and Curtis, 1999; Kearnsand Hunter, 2001; Paerl, 1996; Yamamoto et al., 2011). Sincecolony size strongly influences buoyancy and vertical migration(Kromkamp and Walsby, 1990), the vertical distribution ofcolonies can be affected by colony size and large Microcystis

colonies typically float to water surface, while small colonies (lessthan 36 mm) and single-cell Microcystis showed a nearly uniformvertical distribution over depth (Wu and Kong, 2009).

Bacteria may also affect the colony formation. Shen et al. (2011)have shown that axenic cultures of Microcystis isolated from LakeTaihu became colonial when exposed to bacteria isolated fromLake Taihu. Heterotrophic bacteria also enhanced the aggregationof cultures of Microcystis aeruginosa, most likely through stimula-tion of through mucilage production by M. aeruginosa (Shen et al.,2011). This is supported by previous research, which showsextensive mucilage buildups on the surface of Microcystis cells innaturally occurring blooms (Gao and Yang, 2008; Tien et al., 2002).

Microcystis colony formation may mirror microcystin produc-tion. Microcystis populations are known to be comprised manydifferent strains, from toxic and non-toxic with regard tomicrocystin production (Davis et al., 2009), and this is also thecase in Lake Taihu (Otten et al., 2012). Possibly, high TP and TNconcentration induce the selection of strains that are not able toform colonies and low TP and TN induce the selection of Microcystis

strains able to form colonies. Li et al. (2013) have also suggestedthat under stressful conditions (low light intensities and tem-peratures), Microcystis aeruginosa formed small colonies andexhibited low specific growth rates but standard culture condi-tions yielded single or paired cells with high specific growth rates.

It has been proposed that large colonial algae lose advantagewhen they compete with small colonies or single cell algae whichcan more effectively take up nutrients at low concentrations, andhave less self shading during photosynthesis (Wilson et al., 2010).Microbial miniaturization has been shown to occur during periodsof stress to increase the surface to volume ratio of cells (Morita,1975). However, if nutrients or light are in short supply the abilityto move through a water column and scavenge resources may beadvantageous and this is enhanced as colony size increases andbuoyancy can be controlled. The velocity of movement through thewater is a function of colony size as velocity is proportional to thesquare of the colony radius (Reynolds, 1984).

Colony formation is a complex process influenced by a range ofenvironmental factors. This research has shown that nutrientavailability affected colony size, and this was particularly evidentin the case of P availability. However, exactly what thecyanobacteria are responding to, nutrients or elevated pH, remains

unclear. In nature, the populations are likely to be comprised ofcells/colonies at different ages with different metabolic activities(Brookes et al., 2000). This heterogeneity is likely to lead todifferent growth rates, different ability to acquire resources orproduction of polysaccharides, which will also affect colony size,buoyancy and consequently, bloom formation.

Acknowledgements

We are grateful to all staff in Taihu Laboratory for LakeEcosystem Research (TLLER). This work was supported by theNational Natural Science Foundation of China (40825004,41171368, 41103043, 41271355, 41230744, and 51279194) andUSA National Science Foundation Grants No. ENG/CBET 0826819and 1230543 and DEB 1240851 Dimensions in Biodiversity.[SS]

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author's personal copy...conductivity (ec) in n plus p additions were greater than those...

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