Journal of Applied Phycology https://doi.org/10.1007/s10811-018-1394-5

Effects of different initial pH and irradiance levels on cyanobacterial colonies from Lake Taihu, China Fei Fang 1 & Yan Gao 1 & Lin Gan 1 & Xiaoyun He 2 & Liuyan Yang 1 Received: 29 August 2017 / Revised and accepted: 5 January 2018 # Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Cyanobacteria usually appear in colonies on the surface of lakes, but the microenvironment inside colonies is not as easily detected. An accurate analysis of microenvironment properties within the colonies is key to a better understanding of the formation mechanism of cyanobacterial blooms. To understand the influence of irradiance and pH on the characteristics of cyanobacterial colonies from Lake Taihu, dissolved oxygen (DO) and pH microelectrodes were used to investigate physiological responses within these colonies and in the motionless water blooms at different irradiances and initial pH levels. The results showed that DO and pH increase with increasing irradiance, causing a dynamic alkaline environment to form inside these colonies. The maximum pH varies from 9 to 9.5 at all initial pH readings and the highest DO was achieved in the colonies incubated at an initial pH of 9. The maximum net photosynthesis (Pn) and dark respiratory rate (Rdark) were achieved in the colonies incubated at an initial pH of 8 and 6, respectively. The maximum pH differences were lower in colonies incubated at an initial pH of 10 compared with those incubated at a pH of 6 to 9. Photosynthesis of the colonies raised the aqueous pH to about 10.5, which is similar to the value found inside the colonies. In the motionless water bloom layer, the maximum pH varies from 10 to 10.5 at all initial pH levels and both the highest DO and pH values were achieved at an initial pH of 10. Cyanobacterial photosynthesis first created an alkaline microenvironment in the colonies and then increased the aqueous pH. This elevated aqueous pH promotes photosynthesis of the colonies and further increases the aqueous pH until it is higher than 10. Abundant oxygen bubbles attached at the colonies surface provide extra buoyancy for the colonies. An anaerobic environment forms at 3 to 4 cm depth under the bloom surface, aggravating the outbreak of cyanobacterial bloom. All these physiological characters of microenvironment in cyanobacterial colonies and water blooms favor cyanobacteria as the dominant water bloom species in eutrophic water. Keywords DO . pH . Irradiance . Microenvironment . Cyanobacterial colonies . Cyanobacterial bloom

Introduction Water eutrophication has become one of the most severe problems influencing water quality all over the world. It is often accompanied by massive cyanobacterial blooms, which have become an indicator of water eutrophication (Paerl et al. 2003; Paerl and Otten 2013). Water blooms can disrupt food webs, consume oxygen in the water, and produce microcystins

* Liuyan Yang [email protected] 1

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China

2

Zhejiang Environmental Monitoring Centre, Hangzhou 310012, China

(MCs) which are toxic to other aquatic organisms and water users, such as fish, shellfish, plankton, and humans (Song et al. 2007). They represent a great threat to water quality, ecological stability, and to the economic sustainability of aquatic ecosystems (Paerl and Huisman 2008). Lake Taihu is the third largest freshwater lake in China and has become increasingly eutrophic over the past three decades. The lake is a key drinking water source for the residents who live near it; however, the excessive nutrient loading fueled by human activities has resulted in the annual formation of harmful cyanobacterial blooms (Qin et al. 2010). In 2007, high temperature combined with the hypertrophic conditions resulted in the formation of an extensive bloom composed of cyanobacteria of the genus Microcystis aeruginosa because Microcystis species are capable of producing microcystins. This bloom resulted in serious environmental problems and created a public health emergency (Srivastava et al. 2013; Fu

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et al. 2015). Therefore, a clear understanding of the cyanobacterial bloom formation mechanism is essential for controlling the breakout of cyanobacterial blooms and reducing their negative effects, known as water eutrophication. Cyanobacteria, formerly known as Bblue-green algae,^ are the most notorious bloom formers (Paerl et al. 2001a). Microcystis is the most dominant colonial, bloom-forming genus responsible for harmful, toxic, food-web disrupting, and hypoxia-generating blooms in nutrient-enriched lakes worldwide such as Lake Taihu, China (Paerl et al. 2001b). It represents most of the larger than 20-μm colonies observed in the natural water bodies (Deng et al. 2014a). The cyanobacterial colonies can defend against zooplankton grazing and resist high illumination and temperature damage (Chan et al. 2004). They also display a higher flexibility in nutrient assimilation than the single cell algae and can act to facilitate cyanobacterial dominance (Smith and Kalff 1982). Moreover, cyanobacterial colonies can regulate their vertical movement in the water via a buoyancy regulation mechanism. Cyanobacteria contain gas vacuoles which can regulate the buoyancy to acquire sufficient light and nutrients in the water, allowing cyanobacteria to outcompete other aquatic algae as the dominant bloom species (Reynolds et al. 1987). Other unique adaptations, which allow cyanobacteria to dominate other aquatic algae, is their ability to grow in high temperature and low light (Macedo et al. 2001), capture reduced photosynthetic flux densities, access low concentrations of dissolved CO2, and utilize low ratios of nitrogen to phosphorus (Paerl et al. 2001a). Aqueous pH plays a very important role in the physiological and metabolic activities of cyanobacteria. It strongly influences their cell physiology, colony morphology, and photosynthetic activity (Shruthi and Rajashekhar 2014). The growth of most cyanobacteria is optimal at high pH values between pH 7.5 and 10.0. High pH favors cyanobacterial growth and cyanobacterial bloom formation (Wilson et al. 2010). Cyanobacteria tend to dominate when aqueous pH is elevated (King 1970). The cyanobacterial cell division rate lowers in a low pH environment. Cyanobacterial blooms never occur in acid lakes; thus, even in mildly acidic waters with a pH between 5 and 6, cyanobacteria are uncommon (Kallas and Castenholz 1982). Decreased pH in aquatic bodies will result in non-MC-producing strains, which will outcompete MC-producing strains (Yu et al. 2015). In nature, cyanobacterial photosynthesis can create an alkaline environment which favors its growth and aggravates the breakout of cyanobacterial bloom infestation (Van der Westhuizen and Eloff 1983). The occurrence of high pH in freshwater is common. pH can rise above 9.5 in Lake Taihu when cyanobacterial blooming occurs in summer. Microcystis aeruginosa grows and performs photosynthesis optimally at high irradiance with a temperature above

25 °C, a pH > 8, and excessive phosphorus loading (Bano and Siddiqui 2004). pH has not generally been considered an important determinant of the outbreak of cyanobacterial bloom. Nevertheless, it has been argued that the high pH combined with low CO2—not the high P or low N/P—is the driving force behind cyanobacterial dominance (Hansen 2002; Riding 2006). Most research has focused on unicellular algae in the laboratory or large-scale studies in the field (Kanoshina et al. 2003; Glibert et al. 2004; Hinners et al. 2015). Oinam et al. (2015) studied the effects of pH on the growth of cyanobacteria and competition between cyanobacteria and other phytoplankton in the laboratory. Laboratory studies are helpful, but the in situ approach allows for further exploration of internal microenvironment properties of Microcystis colonies. Such internal activities should be a key factor in the cyanobacterial capacity to withstand adverse environmental conditions such as high pH and high oxidative stress, allowing them to outcompete other algae as the cyanobacterial bloom species. Nevertheless, studies on the physiological characteristics of the internal microenvironment of Microcystis colonies are sparse. We have studied the influence of different temperatures and light intensities on the physiological characteristics of the microenvironment in cyanobacterial colonies (Fang et al. 2014); however, up until the study presented herein, the influence of pH had not yet been studied deeply. Microelectrodes have proven their effectiveness in various fields including biology, medicine, and the environment (Revsbech et al. 1983; Moy et al. 2002; Kaji et al. 2004; Jones et al. 2011). Microelectrodes are ideal tools for in situ measurements without disturbing the structure of samples (Lu et al. 2011). Microelectrodes with a micrometer-scale tip possess various advantages including a high sensitivity, a shorter response time, and a high spatial resolution (Han et al. 2016). Numerous studies have been conducted using microelectrodes. The experimental subjects contain cyanobacterial mats, sludge granules, zooplankton, and biofilms (Epping et al. 1999; Hancke and Glud 2004). Dissolved oxygen (DO), NH4+, NO2−, and H2S profiles in river mats have been measured using microelectrodes (Nakamura et al. 2004). Jørgensen et al. 1985 studied the pH and DO profiles in a planktonic foraminiferan with microelectrodes. Nitrification and denitrification in aerobic nitrifying sludge granules were studied with oxygen and nitrate microelectrodes (Chen et al. 2008). Chiu (2007) studied oxygen diffusion and consumption in active aerobic granules. Therefore, microelectrodes are useful and ideal tools widely used in various fields with great advantages. In this study, pH and DO microelectrodes were used to investigate the influences of different initial pH levels and irradiances on the physiological characteristics of the microenvironment of in-the-field cyanobacterial colonies and water bloom layers sampled from Lake Taihu, to provide some data and theoretical support for recognizing the physiological

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characteristics of the cyanobacterial colonies microenvironment as well as the cyanobacterial water bloom layers and their effect on the formation of cyanobacterial blooms in microscale.

Materials and methods Sampling Water samples contained in-the-field cyanobacterial colonies collected from surface water in the Meiliang Bay area of Lake Taihu in August 2012. At the time of sampling, the in situ water temperature was 32 °C. Cyanobacterial colonies were positively buoyant and accumulated at the lake surface to form a visible cyanobacterial bloom during this period. Macroscopically, the water surface was covered with thick water bloom layers (Fig. 1). The colonies were directly collected from the lake surface using a bucket and immediately transported to the laboratory. The samples were stored at 25 °C under an irradiance of 40 μmol photons m−2 s−1 before the experiment. Microcystis aeruginosa was identified as the main species of the colonies. The diameter of the colonies was concentrated in areas of 0.02 to 0.05 cm. The experiments were conducted within 24 h after sampling.

DO and pH microprofiles in cyanobacterial colonies at different initial pH and irradiances Granules with green, intact, and round structures with a diameter of 2 ± 0.1 mm were selected for the experiments (Fig. 2), and the colonies were immobilized on a nylon mesh using a wide-mouth pipette (Fig. 3). The nylon mesh was fixed on a small glass beaker (10 mL) which was inserted into a larger one (50 mL). BG-11 medium adjusted to different pH values (6, 7, 8, 9, and 10) was added to the beaker and the colonies were kept in the media with a pH of 6, 7, 8, 9, and 10, respectively. The

Fig. 1 Cyanobacterial bloom in Meiliang Bay of Lake Taihu

Fig. 2 Cyanobacterial colonies sampled from Lake Taihu embedded in etamine pores for microelectrode measurement

pH was adjusted with 0.1 M NaOH and HCl to obtain the given pH. Afterwards, the experimental colonies were preincubated in the dark overnight at 25 °C. The next morning, after incubation in the dark for 12 h, DO and pH microprofiles of the colonies incubated in the medium with an initial pH of 6 were measured in the dark at 25 °C. Then, irradiances were set to 20, 40, 80, and 120 μmol photons m−2 s−1, respectively, and each steady-state DO and pH microprofile was measured after incubation for about 1 h. Steady-state gradients of pH and oxygen were achieved after about 30 min of light exposure. Afterwards, the procedure was followed in the colonies incubated in media with an initial pH of 7, 8, 9, and 10, respectively. Three experimental colonies were preincubated at an initial pH of 6, 7, 8, 9, and 10, respectively. Each colony was measured at least three times. The final DO and pH value was the average value of the three measurements with the standard deviation of the mean value. Experiments on cyanobacterial colonies were performed under a dissection microscope to position the tip of the electrode at the colony surface. The surface of the colonies was regarded as the reference depth for vertical positioning. Experiments were conducted from the overlying water to the interior of cyanobacterial colonies in intervals of 100 μm. Microelectrodes were propelled downward automatically via a motor-driven micromanipulator (Unisense, Denmark). The samples were illuminated with a 150-W fiber-optic tungsten halogen light (Schott KL 1500) with neutral density filters inserted in the light path to reduce incident irradiance. The irradiance (400–700 nm) was measured with a quantum irradiance meter (Waltz). Cyanobacteria usually grow well at 25 °C and 40 μmol photons m−2 s−1. Too much irradiance and high temperature restrict the cyanobacterial photosynthesis (Fang et al. 2014). The outbreak of cyanobacterial blooms makes the water pH undergo great change in Lake Taihu. Therefore, the five different pH values and illumination intensities were chosen as experimental conditions and the experimental temperature was set to 25 °C.

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Fig. 3 Micrographs of cyanobacterial colonies with different sizes (left, middle) and the intracellular space in cyanobacterial colonies (right)

DO and pH microprofiles in motionless water bloom layers at different initial pH values and irradiances

Calculation of net photosynthesis and dark respiration

Water samples containing cyanobacterial colonies were put into deionized water three times and resuspended to remove impurities. Afterwards, the equivalent volume of cyanobacterial blooms was added to a 50-mL BG-11 medium, and the aqueous pH was set to 6, 7, 8, 9, and 10, respectively. The pH was adjusted with 0.1 M NaOH and HCl. The thickness of the water blooms was about 0.1 cm in a motionless water body. Then, the blooming water body was preincubated in the dark overnight at 25 °C. The following morning, DO and pH profiles in the bloom water layer with the initial pH of 6 were measured in the dark at 25 °C. Afterwards, irradiance levels were set to 20, 40, 80, and 120 μmol photons m−2 s−1, and each steady-state DO and pH profiles were measured after incubation for about 1 h. Steady-state gradients of pH and oxygen in the water blooms were achieved after about 30 min of light exposure. Then, the procedure was followed by steady-state measurements in the medium with an initial pH of 7, 8, 9, and 10 in that order. Measurements were repeated three times for each initial pH condition at different positions and the final DO and pH values were the average value of three series of measurements with the standard deviation of the mean value. DO microprofiles were measured with a Clark-type oxygen microelectrode (Unisense A/S, Denmark) with a tip diameter of 25 μm, a stirring sensitivity of < 1%, and a 90% response time of < 1 s. Linear calibration was done in an air-saturated BG-11 medium and an anaerobic solution. pH microprofiles were measured with an Ag–AgCl microelectrode (Unisense, Denmark) with a tip diameter of 25 μm in combination with an Ag–AgCl reference electrode. A linear calibration of the pH electrode was done with NBS buffers (Mettler Toledo, pH 4, 6.86, and 9.2). The typical electromotive force was 55 mV pH −1, and 90% response time was < 1 s. The output current of the microelectrodes was recorded by a Microsensor Multimeter (Unisense, Denmark) connected to a personal computer. The signals from the sensors were transformed to DO concentration in micromoles per liter. All calibrations were conducted at 25 °C.

DO microprofiles in the cyanobacterial colonies were the comprehensive result of photosynthesis, respiration, and oxygen diffusion. Diffusion rate of DO levels across the water– colony interface was calculated by Fick’s first law of diffusion: J0 = −фD0 dC/dz, assuming a uniform depth distribution of porosity ф and a water diffusion coefficient D0. C represents the DO concentration. Depth, z, represents the depth coordinate, which was scaled from 0 at the water– cyanobacteria interface, and then scaled positively downward. D0 is the free solution molecular diffusion coefficient of dissolved oxygen in water, corrected for salinity and temperature. D0 in water was 2.06 × 10−9 m−2 s−1 at 25 °C (Broecker and Peng 1974). dC/dz was determined from the linear section of DO concentration gradient in the diffusion boundary layer (DBL) of the colonies. J0 is positive on the assumption that a dissolved oxygen flux was directed from the water to the colonies. Pn and Rdark represent net photosynthesis and dark respiration. Pn was estimated as the flux of oxygen from the colonies to the surrounding water across the DBL calculated from Pn = Jup = − D0 dC/dz. Dark respiration, Rdark, was calculated as the flux of oxygen from the water to the colonies calculated from Rdark = Jdown = − D0 dC/dz (Kühl et al. 1996; Lassen et al. 1998).

Results DO microprofiles in cyanobacterial colonies at different initial pH conditions DO microprofiles of cyanobacterial colonies incubated at different initial pH conditions were measured under different irradiances (0–120 μmol photons m−2 s−1) (Fig. 4). Oxygen diffusion between the colonies and the surrounding water was limited by the DBL around the colonies. DO concentrations increased from the surrounding water to the interior of cyanobacterial colonies and achieved the maximum concentration in the core of the colonies. DO concentrations

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Fig. 4 DO microprofiles in cyanobacterial colonies incubated at different irradiances (μmol photons m−2 s−1) and different initial external pH of 6 (a), 7 (b), 8 (c), 9 (d), and 10 (e) (n = 3)

increased with increasing irradiance except for a decrease occurring at 120 μmol photons m−2 s−1 in the colonies incubated

at an initial pH of 6. The maximal DO concentration in the core of the colonies increased from 532 μmol L−1 at an initial

J Appl Phycol Table 1

J0 in the diffusive boundary layer of the cyanobacteria–water interface (n = 3)

Illumination/μmol photons m−2 s−1 0

pH 6

pH 7

pH 8

pH 9

pH 10

0.124 ± 0.012

0.089 ± 0.011

0.050 ± 0.013

0.100 ± 0.006

0.037 ± 0.006

20

− 0.338 ± 0.005

− 0.071 ± 0.012

− 0.320 ± 0.006

− 0.178 ± 0.008

− 0.225 ± 0.013

40 80

− 0.604 ± 0.011 − 0.558 ± 0.022

− 0.364 ± 0.013 − 0.888 ± 0.016

− 0.681 ± 0.012 − 1.066 ± 0.014

− 0.545 ± 0.002 − 0.639 ± 0.005

− 0.511 ± 0.009 − 1.234 ± 0.011

120

− 0.320 ± 0.012

− 0.838 ± 0.011

− 1.441 ± 0.011

− 1.313 ± 0.004

− 1.254 ± 0.012

pH of 6 to 630 μmol L−1 at an initial pH of 7 and further increased to 718 μmol L−1 as the initial pH increased to 8, and it continued to increase to 974 μmol L−1 at pH 9 and then decreased to 850 μmol L−1 as the initial pH increased to 10. Thus, the maximal DO concentration was achieved in the core of the colonies incubated at an initial pH 9. It increased with rising external initial pH from 6 to 9 but decreased when achieving a pH of 10. The lowest maximal DO level was achieved in the core of the colonies incubated at the external pH 6. Therefore, acid and strong alkaline environment will inhibit the photosynthesis of the colonies.

DO diffusion rate at the cyanobacteria–water interface The DO flux at the cyanobacteria–water interface J0 was calculated by Fick’s first law of diffusion. It was positive when oxygen was diffused from the surrounding water to the colonies. As seen from Table 1, J0 was positive in the darkness and became negative in bright light under all initial pH conditions. Similar variations of J0 were observed in the colonies incubated at initial pH 8–10. J0 increased with rising of irradiance and the maximum J0 was achieved at an initial pH of 8. As seen from Fig. 5, Pn increased with an increase of irradiance except when a decrease appeared at 40 μmol photons m−2 s−1 at an

initial pH of 6 and 120 μmol photons m−2 s−1 at an initial pH of 7. The lowest Pn was achieved at pH 6, and the highest Pn was achieved at pH 8. The highest Rdark was achieved at pH 6, and the lowest Rdark was achieved at pH 10. Therefore, slight alkaline and acid conditions benefited the photosynthesis and the respiration of the colonies, respectively.

pH microprofiles in cyanobacterial colonies under different initial pH conditions pH microprofiles of the colonies under different pH and irradiances are shown in Fig. 6. Like the DO microprofiles, the pH increased from the surrounding water to the interior of the colonies and achieved the maximum value in their core. The pH change became apparent when it turned from dark to light. The maximal difference in pH values between darkness and the brightest light could be up to 2.5 in the granules incubated at an initial pH of 6. At an initial pH of 6, the change in pH was not apparent as the irradiance increased to higher than 40 μmol photons m−2 s−1, and the maximum pH achieved was 9.20 at 120 μmol photons m−2 s−1. At an initial pH of 7, the maximum pH gradually increased with an increase of irradiance, and the maximum pH achieved was 9.55 at 120 μmol photons m−2 s−1. At an initial pH of 8, the maximum pH at 80 μmol photons m−2 s−1 was similar to that measured at

Fig. 5 Pn (left) and Rdark (right) in the colonies incubated at different initial external pH values and irradiances (μmol photons m−2 s−1) (n = 3)

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Fig. 6 pH microprofiles in cyanobacterial colonies incubated at different irradiances (μmol photons m−2 s−1) and different initial external pH values of 6 (a), 7 (b), 8 (c), 9 (d), and 10 (e) (n = 3)

120 μmol photons m−2 s−1, and the maximum pH value was 9.45 at 120 μmol photons m−2 s−1. At pH 9, the change of pH

in the light was not as pronounced as those displayed at pH 6– 8 and the maximum was 9.71 at 120 μmol photons m−2 s−1.

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The difference in maximum pH between the surrounding water and the interior of the colonies became less (< 0.5) as the external initial pH increased to 10 and the maximum pH in the core was 9.80. The lowest maximum pH was achieved in the colonies incubated at pH 6, but the change of pH was the most apparent. The highest maximum pH was achieved in the colonies incubated at pH 10, but the change of pH was the most inconspicuous. The difference of the maximum pH between darkness and the highest light was 2.74, 2.70, 2.35, 1.01, and 0.48 at pH 6, 7, 8, 9, and 10, respectively. It decreased with the external initial pH increase from 6 to 10. The difference of the maximum DO between darkness and the highest light was 364, 493, 577, 834, and 696 μmol L−1 at pH 6, 7, 8, 9, and 10, respectively. It increased with the external initial pH increase from 6 to 9 but decreased as the external initial pH increased to 10 (Fig. 7).

DO microprofiles in motionless cyanobacterial bloom water layer at different initial pH values Similar to the experimental measurements in the colonies, DO concentration in a motionless cyanobacterial blooming water layer increased with an increase of irradiance (Fig. 8). In the light, DO level achieved its maximum rate at the water surface or increased from the water surface and achieved the maximum under the water surface and then decreased with the increase of water depth. Afterwards, an anaerobic environment was formed at a depth of 3–4 cm under the water surface. At an initial pH of 6, the maximum DO level was achieved at the water surface at 40 μmol photons m−2 s−1. The maximum DO concentration at 80 μmol photons m−2 s−1 was similar to the 431 μmol L−1 measured at 120 μmol photons m−2 s−1. At an initial pH of 7, the maximum DO level was achieved at the water surface at 80 μmol photons m−2 s−1 and the maximum

value reached 409 μmol L−1 at 120 μmol photons m−2 s−1. Both maximum DO levels at pH 8 and 9 were achieved at the water surface at 120 μmol photons m−2 s−1, and the value was 439 and 493 μmol L−1, respectively. At an initial pH of 10, the DO concentration gradually increased with rising irradiance, and the maximum level was achieved at 653 μmol L−1 at 120 μmol photons m−2 s−1. Different from the maximum DO level in the colonies, the highest DO in the motionless cyanobacterial bloom layer achieved at an initial pH of 10.

pH microprofiles in motionless cyanobacterial bloom water layer at different initial pH values pH microprofiles in cyanobacterial bloom layers at different initial pH and irradiances are shown in Fig. 9. Similar trends were observed in changes of the pH in the cyanobacterial bloom layer. The pH increased and achieved its maximum value at the water surface or underwater and then decreased to a relatively stable low value in deep water. At an initial pH of 6, the change of pH was not as pronounced as the irradiance increasing from 40 to 120 μmol photons m−2 s−1, and the maximum pH was 10.02 at 80 μmol photons m−2 s−1. At pH 7, the pH in the upper part of the water increased to 7.5 after an overnight culture in darkness. The change of pH was not as pronounced as the irradiance increased from 80 to 120 μmol photons m−2 s−1, and the maximum pH reached 10.42 at 80 μmol photons m−2 s−1. At initial pH of 8, 9, and 10, all the pH values gradually increased with the increase of irradiance and the maximum pH values reached 10.23, 10.48, and 10.52 at 120 μmol photons m−2 s−1, respectively. Similar to the maximum DO concentration, the maximum pH in the motionless water bloom layer reached at an initial pH 10 value.

Fig. 7 Difference of pH (left) and DO (right) between darkness and different irradiances (μmol photons m−2 s−1) in the core of cyanobacterial colonies incubated at different initial external pH values (n = 3)

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Fig. 8 DO microprofiles in cyanobacterial bloom water layers at different irradiances (μmol photons m−2 s−1) and different initial pH values of 6 (a), 7 (b), 8 (c), 9 (d), and 10 (e)

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Fig. 9 pH microprofiles in cyanobacterial bloom water layers at different irradiances (μmol photons m−2 s−1) and different initial pH values of 6 (a), 7 (b), 8 (c), 9 (d), and 10 (e) (n = 3)

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Discussion Cyanobacterial blooms have become one of the world’s most serious environmental problems in recent years (Bowling and Baker 1996; Ha et al. 1998; Sorichetti et al. 2014). Cyanobacteria are the most common freshwater harmful algal taxa. They can proliferate themselves in many ways and accumulate at the water surface to become a macroscopic water bloom (Mur et al. 1999). The decay of cyanobacteria can produce odoriferous and unsightly scum, resulting in hypoxia and anoxia of underlying waters, which may lead to fish and bottom fauna mortalities. In addition, some bloom-forming species such as M. aeruginosa are capable of producing MCs, severely threatening the water safety and human health (Paerl 1996). Therefore, a study on the formation mechanism of water bloom is of great significance for cyanobacterial bloom control. Cyanobacterial blooms regularly occur in the surface waters of Lake Taihu, China, and the main bloom-forming species is M. aeruginosa (Xu et al. 2010; Deng et al. 2014b). A long evolutionary history of cyanobacteria has endowed them with a series of morphological, physiological, and ecological adaptations in response to various environmental conditions (Paerl et al. 2001b; Oliver and Ganf 2002). Microcystis aeruginosa usually exist in colonial aggregates in the field, creating a microenvironment within the colonies, which can defend against zooplankton grazing and resist extreme environmental damage such as high temperature and high irradiance (Brock 1985; Wang et al. 2010). Furthermore, colonial M. aeruginosa can regulate their vertical movement via buoyancy regulation to rapidly migrate between radiance-rich surface waters and nutrient-rich deeper waters, thereby satisfying both light and nutrient requirements (Deng et al. 2014a). An accurate detection of the properties of the microenvironment inside and outside the colonies should be very important for a better understanding of the formation mechanism of cyanobacterial bloom. Nevertheless, the properties of the microenvironment in such colonies and the effects of environmental factors are sparse. pH is one of the key biogeochemical parameters indicating the thermodynamic state of the acidbased process and overall balances between multiple reactions and transport processes within natural environments (Yu et al. 2015). It has been argued that the low CO2 or high pH is the driving force behind cyanobacterial dominance (Kanoshina et al. 2003; McCarthy et al. 2007). We studied the influence of irradiance and temperature on the photosynthetic activity of the cyanobacterial colonies (Fang et al. 2014), but the information of the influence of pH on the microenvironment in the cyanobacterial colonies was not clear. DO and pH microprofiles in the colonies and motionless cyanobacterial bloom water layers were studied at different initial pH values (6–10) and irradiances using DO and pH microelectrodes. The results showed that a dynamic microenvironment was created both in the colonies and in the water

bloom layer. In the colonies, the maximum DO concentration increased with rising external pH from 6 to 9 but decreased when reaching a pH of 10. The highest DO level reached was approximately 1000 μmol L−1 in the core of the colonies incubated at an initial pH of 9. The change of pH was apparent in the colonies incubated in the medium with an initial pH of 6 and 7, but it became inconspicuous when the external pH value increased above 9. The maximum pH differences were lower in the colonies incubated at an initial pH 10 compared with those incubated at an initial pH from 6 to 9. This revealed that slight alkaline conditions favor cyanobacterial photosynthesis. Nevertheless, it was inhibited when the external pH was too high (Pedersen and Hansen 2003a). An initial external pH of 9 is the most appropriate condition for cyanobacterial photosynthesis. Similar to the value at an initial pH of 9, the highest pH in the colonies’ core (incubated at an initial pH 10) is still lower than 10 as the external pH increases one unit. This indicates that a relatively stable internal microenvironment was created in the colonies to resist the variation of external pH change. Colonies can create a relatively high alkaline microenvironment (pH 8–9) from within when the external water pH is low (6–8); nevertheless, an internal pH below 10 is created when the external pH exceeds 10. Numerous studies have been conducted on the influence of pH on the growth, enzyme activity, and physiology of cyanobacteria (Dokulil and Teubner 2000; Menéndez and Comin 2001; von Sperling et al. 2008). The study of five marine cyanobacterial species showed that all cyanobacterial species appear to prefer near-neutral to alkaline pH. The optimal growth pH of most cyanobacteria is 7 to 10 pH (Leavitt et al. 1999). The growth of Synechocystis increased with rising pH, but it was significantly repressed at both high (pH 11) and low pH (pH 6). The pH varied between 7.4 and 9.0—between darkness and saturating light intensities in aggregates formed by Aphanizomenon sp. and Nostoc spumigena from the Baltic Sea. It is suggested that the close association of heterotrophic and autotrophic organisms creates a pH microenvironment that is beneficial to iron uptake for the cyanobacteria, which in turn may release surplus nutrients to the heterotrophic community (Ploug 2008). Several possible mechanisms whereby high pH can benefit the cyanobacterial growth include high pH optima for nutrient uptake enzymes in cyanobacteria, decreases in metal solubility at high pH, and uptakes of different forms of phosphate (Keithellakpam et al. 2015). Meanwhile, abundant proteins are differentially expressed under high pH stress, and some proteins only appear in the high-pH-stressed cells, which include photosynthesis and respiration proteins (Ma et al. 2014). Furthermore, an intracellular nitrogen supply can also be affected by high pH stress treatment; moreover, high pH may interrupt the nitrogen and carbon uptake pathways in cyanobacteria (Zhang et al. 2009). Therefore, an alkaline microenvironment created inside the colonies in the light can benefit the nutrient uptake and photosynthesis of

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the colonies, which is a key factor in cyanobacteria outcompeting other algae to be the bloom-dominant species. Jørgensen et al. (1985) found that DO at the foraminifera shell surface was lowered to 50% air saturation; meanwhile, the pH was lowered from 8.23 to 8.15 in the darkness. On the contrary, DO increased several times which was higher than the air saturation level, and pH increased in the light. In the present study, the results show that the colonies are surrounded by a thick boundary film of the water body which has a chemical composition highly different from that of the bulk lake water body. DO and pH increase with increasing irradiance, but a decrease occurs in the colonies incubated in a medium with an initial pH of 6 at 120 μmol photons m −2 s −1. Photoinhibition occurs in colonies incubated at a weak acidic medium and high irradiance; nevertheless, high pH combined with high irradiance does not result in a decrease of DO production. Photoprotective carotenoids can prevent the cyanobacteria from photodamage and ensure long-time survival under high irradiance (Miller et al. 1998). High pH benefits cyanobacterial phycobiliprotein production and ammonium excretion, thereby enabling cyanobacteria to utilize light more efficiently (Keithellakpam et al. 2015). Cyanobacteria exposed to high irradiance combined with low external pH may be damaged by photooxidation, and photorespiration results in a decrease of DO concentration of the cyanobacterial colonies (Miller et al. 1998; Li et al. 2013). Colonial cyanobacteria can protect the interior algae from photodamage. Appropriate irradiance favors cyanobacterial photosynthesis and growth, which increases the bloom outbreak. Buoyancy regulation enables cyanobacterial colonies to acquire sufficient light at the water surface and accumulate as visible water blooms (Ibelings 1996). There cyanobacteria can take advantage of bright light and shade and other phytoplankton which can strengthen their competitiveness (Zhang et al. 2011). Visible oxygen bubbles released from the colonies provide extra buoyancy of the colonies (Fig. 10). Nevertheless, after a long-term exposure to bright light intensity, decreased oxygen production combined with increased carbohydrate production will reduce the buoyancy of the colonies and make them move downwards to avoid

Fig. 10 Cyanobacterial bloom surface filled with oxygen bubbles

photodamage (Deacon and Walsby 1990; Baroli and Melis 1998). Oxygen production in the colonies is the comprehensive result of photosynthesis, respiration, and oxygen diffusion (Huang and Chen 2013). The net photosynthesis Pn and dark respiration Rdark of the colonies has been calculated by Fick’s first law. A positive value of J0 indicates that oxygen diffuses from the water to the colonies, and a negative one means the opposite. The thickness of DBL is determined from the slope of the DO profile, which is the largest radial distance yielding the same total flux in the water as that found at the surface of the colonies (Rasmussen and Jørgensen 1992). In the present study, the fluxes were positive in the darkness and became negative in the light (Table 1). Pn increased with rising irradiance except a decrease appeared in the colonies incubated in the medium with an initial pH of 6 and 7 at 120 μmol photons m−2 s−1. The highest Pn and Rdark was achieved in the medium with an initial pH of 8 and 6, respectively. This indicates that oxygen diffused from the colonies to the surrounding water at high irradiance; thus, a slight alkaline condition favored the cyanobacterial photosynthesis, while the acidic condition promoted the cyanobacterial respiration. In Aphanizomenon sp. and N. spumigena aggregates from Baltic Sea, net oxygen fluxes at the air–sea interface were 2.7-fold higher than those at the aggregate–water interface beneath the layers. Pn at light saturation was 1.7–2.4 mmol O2 m−2 h−1, and Rdark varied between 0.21 and 0.50 mmol O2 m−2 h−1 (Ploug 2008). In the proteomic analysis of plasma membranes of cyanobacterium Synechocystis sp. in response to high pH stress, hundreds of genes were upregulated and many proteins including transport proteins and proteins were involved in cell division. Photosynthesis and respiration changed in abundance at high pH stress. Several subunits of photosystems and the respiratory chain mediated by cytochrome c oxidase were clearly enhanced under high pH stress condition (Zhang et al. 2009). It may also be the reason for the change of photosynthetic and respiration rate at high pH stress in this study. In summer, a cyanobacterial bloom dominated by M. aeruginosa covered Meiliang Bay, one of Taihu’s main bays (Kong et al. 2009; Fu et al. 2015). They had created a thick cyanobacterial bloom layer at the lake surface where the water pH maintains at 7.5 to 9.5 and the highest pH can achieve 10.5 (Shi et al. 2016). In our study of the effect of pH on the water blooms from Lake Taihu, we found that the DO production in the water blooms increased with rising initial pH levels, achieving the maximal values at pH 10, and all the maximum pH in the water was maintained at 10 to 10.25 pH regardless of the initial pH levels. Combined with the measurements in colonies, it was determined that cyanobacterial photosynthesis increased the DO and pH levels in the colonies and then elevated the values in the water body. Elevated aqueous pH in turn enhances the photosynthesis of the cyanobacterial colonies and further increases the aqueous pH; nevertheless, both the DO

J Appl Phycol

production and pH levels in and out of the colonies stopped increasing when the aqueous pH reached 10 (Fig. 11). This is a very important self-regulation mechanism of cyanobacterial colonies, which can keep a continued proliferation of cyanobacteria and eventually form harmful blooms. It revealed that cyanobacteria have the ability to keep photosynthesis at high pH stress and to effectively utilize bicarbonate; these properties favor them in bloom conditions. King (1970) showed that cyanobacteria tend to be dominant when CO2 is low and pH is elevated. Higher pH can induce greater cyanobacterial dominance under identical nutrient loading. Cyanobacterial dominance declines when pH is reduced by increased respiration or upwelling of hypolimnetic CO 2 (Jensen et al. 1994). Cyanobacterial variation is more closely related to pH than to CO2. The pH alteration is more important than the other direct impacts (Paerl 1983; Caraco and Miller 1998). Elevated pH (> 9) in a natural water body will influence the entire plankton community mainly by reducing the species richness and by inducing cyanobacterial blooming due to the loss of grazing (Pedersen and Hansen 2003a, b). Meanwhile, pH affects cyanobacterial dominance by impacting their removal rate by cyanophages, which can have low pH optima for their growth. Therefore, a relatively stable high pH microenvironment created in the water body benefits the photosynthesis of cyanobacterial colonies and aggravates the outbreak of cyanobacterial bloom. Many cyanobacterial blooms accumulate at the water surface, where light is sufficient (Cai and Kong 2013). Rapid

bloom development can result if the division and growth rates are fast, as is the case of buoyant species such as M. aeruginosa, when stable water column conditions favor surface accumulations (Steinberg and Hartmann 1988; Yuan et al. 2008; Zhang et al. 2011). Accumulations may result in an extremely high water DO level at the bloom surface (Fabbro and Duivenvoorden 1996; Smith 2003). Reactive byproducts of oxygen, such as superoxide anion radical (O2−), hydrogen peroxide (H2O2), and the highly reactive hydroxyl radical (OH−), are produced in cells grown aerobically (Cabiscol et al. 2000). Oxidative stress arises when the level of active oxygen increases to a level that exceeds the cell’s defense capacity. Cyanobacteria can build up mechanisms to protect themselves against oxidative stress, with enzymes such as catalase and superoxide dismutase (Warhurst 2014). After rapid proliferation, cyanobacteria may rapidly consume nutrients, deplete inorganic carbon supplies, increase turbidity, and thus cause a sudden decrease in biomass. This usually accompanies decaying and odoriferous scum (Paerl et al. 2001a, b). Scum can rob the oxygen in the underlying waters and result in significant biological and physicochemical changes of the water body, including hypoxia or anoxia environment, which is fatal to most fish and shellfish, as well as release of nutrients from sediments, which further aggregates cyanobacterial blooms (Qin et al. 2015). In Lake Taihu, the observed water DO concentration was 6 mg L−1 in the most serious cyanobacterial bloom bays in August. However, DO was 12 mg L−1 in winter (Wu et al. 2015). During a windless summer day, a hypoxia

Fig. 11 Interaction of microenvironment in the cyanobacterial colonies and water bloom layers and the pH regulation mechanism of the colonies

Cyanobacterial photosynthesis increasing the water pH; Respiration decreasing the water pH

Float up

Oxygen bubbles Photosynthesis

Cyanobacterial colonies

Microenvironment

Water bloom layer

in the colonies

microenvironment Slight alkaline water (pH 8-9) favoring cyanobacterial photosynthesis, Strong alkaline water inhibiting photosynthesis (pH >10)

Hypoxia environment (3-4 cm depth under the water surface)

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environment forms in the underlying waters of cyanobacterial blooms in Lake Taihu, and the cyanobacterial decaying and DO stratification can aggravate such phenomenon (Zhang et al. 2008). In the present study, DO achieved the maximum near the water surface and decreased to 0 μmol L−1 at 3–4 cm depth under the cyanobacterial bloom surface and then an anaerobic environment forms in the underlying water. A hypoxia environment forms in the underlying water, which can further aggravate or induce the outbreak of cyanobacterial blooms (Li et al. 2014; Xu et al. 2015). pH-dependent changes in phytoplankton growth rate may actually be due to dissolved inorganic carbon speciation and concentration (Ibelings and Maberly 1998; Huertas et al. 2000). Taraldsvik and Myklestad (2000) revealed that some species are unable to maintain a constant internal pH when the external medium pH varies. A change in the extracellular pH will affect not only the growth rate but also the physiochemical activity of the cells (Shapiro 1997). Nevertheless, a relatively stable alkaline environment is generated in the cyanobacterial colonies regardless of how much the external pH varies in the light. The maximum pH in the core of the colonies can immediately increase to a high level (pH 8 and 9) in sunlight despite the water pH maintaining a low level (pH 6 and 7); furthermore, it maintains a pH of approximately 9.5 even when the external aqueous pH increases from 9 to 10. Meanwhile, the maximum external aqueous pH outside of the colonies stops increasing and maintains a pH lower than 10.5 in the light. All these can provide an optimum microenvironment for the physiochemical activity of the cyanobacterial colonies, which enhances cyanobacterial competitiveness with other algae. Decreased CO2 availability (elevated pH) can result in MC-producing strains such as M. aeruginosa to dominate non-MC-producing strains (Tillett et al. 2000). Growth of cyanobacteria is affected by the equilibrium of the inorganic carbonate species present, and their uptake into the cells, including CO 2 , HCO 3 − , and CO 3 2− (Shapiro 1990; Taraldsvik and Myklestad 2000; Beardall and Raven 2016). The preference is for CO2 over HCO3− as a photosynthetic carbon source, but there is little free CO2 in water when pH exceeds 10 (Gavis and Ferguson 1975; Paerl and Ustach 1982). Colony formation is a trade-off between diffusion of gases through the colony, light requirement, buoyancy regulation, predator avoidance, and nutrient uptake (Yamamoto et al. 2011). It has been proposed that a large colonial cyanobacteria loses their advantage when they compete with small colonies which can more effectively take up nutrients at low concentrations. Thus, an increase of water pH and carbon limitation can generate smaller M. aeruginosa colonies (Ma et al. 2014). Due to the small size of the cyanobacterial colonies, the molecular diffusion between the colonies and surrounding water is rapid (Yang et al. 2008), which causes rapid changes in DO and pH microprofiles in the colonies in response to external environmental variations. This activity

creates a microenvironment that benefits from the physiology activity of cyanobacterial colonies. pH is a key environmental factor that influences the physicochemical characteristics of cyanobacterial colonies in lakes with cyanobacterial blooms such as in Lake Taihu. The physicochemical activities of the cyanobacterial colonies are controlled by both internal pH and external pH. A stable alkaline and high DO microenvironment is formed within the cyanobacterial colonies in sunlight. Photosynthesis of the cyanobacteria first raises the pH in the colonies and then increases the aqueous pH until it reaches approximately 10.5, a similar value to that found inside the colonies. The microenvironment formed in the colonies is a key source of the cyanobacteria blooms’ capacity to be the dominant water bloom species. Acknowledgments This work has been supported by grants from the National Special Program of Water Environment (2017ZX07204) and National Basic Research Program of China (2008CB418102).

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