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Dynamics of microcystin production and quantification ofpotentially toxigenic Microcystis sp. using real-time PCR

Ankita Srivastava a, Gang-Guk Choi b, Chi-Yong Ahn b, Hee-Mock Oh b, Alok Kumar Ravi c,Ravi Kumar Asthana a,*aCentre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221 005, IndiabEnvironmental Biotechnology Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon, Republic of KoreacDepartment of Ocular Pharmacology and Pharmacy, Dr. R.P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences,

New Delhi 110 029, India

a r t i c l e i n f o

Article history:

Received 18 November 2011

Accepted 19 November 2011

Available online 27 November 2011

Keywords:

LCeMS

Microcystis sp.

Microcystin

Real-time PCR

* Corresponding author. Department of Bota0542 2368174.

E-mail address: asthana.ravi@gmail.com0043-1354/$ e see front matter ª 2011 Elsevdoi:10.1016/j.watres.2011.11.056

a b s t r a c t

Cyanobacterial blooms in eutrophied water body are generally composed of various

genotypes with or without microcystin-producing genes (mcy gene cluster). Thus there is

a need for quantification of potent toxin producing strains. The present study aimed at

identifying microcystin variants and its producer strains in Durgakund pond, Varanasi,

India, based on quantification of cpcBA-IGS and mcyA (condensation domain) genes using

real-time PCR and LCeMS. Increase in microcystin concentrations was correlated with

increase in mcyA copy number and the level of pigments (chlorophyll a, phycocyanin and

carotenoids). Also, selected environmental factors (water temperature, light irradiance,

rainfall, pH, N and P) and the concentration of microcystin variants (MC-LR, -RR and -YR)

were also assessed in samples during May 2010 to April 2011 to establish the possible

correlation among these parameters. Nutrients favored cyanobacterial bloom but it could

not be correlated with the levels of microcystin variants and seemed to be geographically

specific. Microcystis sp. dominant in the pond comprised potentially toxigenic cells. The

ratio of potentially toxigenic Microcystis sp. to that of total Microcystis sp. ranged from 0% to

14%. Such studies paved the way to identify and quantify the most potent microcystin

producer in the tropical aquatic body.

ª 2011 Elsevier Ltd. All rights reserved.

1. Introduction Along with the environmental factors, bloom composition

Cyanobacterial mass proliferation constitutes serious threat

to the water quality in many freshwater bodies worldwide as

they synthesize structurally and functionally unrelated but

highly potent toxins (Carmichael, 1997). Microcystins in

aquatic bodies result from cyanobacteria such as Microcystis,

Anabaena, Nostoc and Oscillatoria endowed with the micro-

cystin synthetase gene cluster (Nishizawa et al., 2000).

ny, Banaras Hindu Unive

(R.K. Asthana).ier Ltd. All rights reserved

plays a pivotal role in toxicity prediction as microcystin-

producing cell quota may vary up to 3e4 orders of magni-

tude (Blackburn et al., 1997; Carmichael, 1997). The abundance

of toxic and non-toxic genotypes within the algal bloom varies

with habitats as well as the environmental regimes. It is here

thatmolecular approaches allow the detection of specific DNA

sequences to distinguish between the toxic and non-toxic

strains of Microcystis (Baker et al., 2002). PCR amplification of

rsity, Varanasi 221 005, India. Tel.: þ91 0542 2307146/47; fax: þ91

.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7818

variousmcy genes has been used for an early detection of toxic

Microcystis blooms (Neilan et al., 1999; Kim et al., 2010).

India too has a number of large reservoirs and rivers with

toxic blooms and microcystin variants as documented by

many (Prakash et al., 2009; Sangolkar et al., 2009). The uneven

distribution of toxic and non-toxic strains within most genera

prevented accurate diagnosis of bloom samples (Pearson and

Neilan, 2008). Although conventional PCR approach based on

amplification of mcy genes was used to identify toxic cyano-

bacterial species in various water bodies (Ghosh et al., 2008;

Sangolkar et al., 2009; Kumar et al., 2011), the quantification

of potentially toxigenic cyanobacterial strains using real-time

PCR is almost lacking in the Indian context. Kumar et al. (2011)

reported the presence of hepatotoxigenic Microcystis sp. from

Durgakund pond but it never accounted for the abundance or

the dominance. The perennial Microcystis sp. in the pond has

important implications in the health risk of a large number of

people using such waters for religious purposes every day.

Studies on toxic blooms in other countries already involved

real-time PCR to quantify mcy genes and to monitor

microcystin-producing cyanobacteria (Kurmayer and

Kutzenberger, 2003; Vaitomaa et al., 2003; Rinta-Kanto et al.,

2005; Kim et al., 2010; Al-Tebrineh et al., 2011). Microcystis sp.

dominance is prevalent in the toxic blooms of temperate,

freshwater environments (Baxa et al., 2010). Thus an in depth

study on trends in dominance and peak of abundance of

Microcystis and the presence of different microcystin variants

in tropical regions is needed to mediate the control of toxic

blooms.

Light, temperature, nutrients (mainly N and P) and trace

metals affect bloom toxicity by direct stimulations of cell

division and growth (Sivonen, 1990; Oh et al., 2000; Davis et al.,

2009). However, Utkilen and Gjølme (1995) observed no effect

of nitrate and phosphate limited conditions on toxin produc-

tion in Microcystis aeruginosa. It is here that our study focused

at the real-time PCR approach to recount for the abundance of

total Microcystis sp. using cpcBA-IGS and the potentially toxi-

genic cyanobacteria using mcyA condensation domain for

monitoring the toxicity of the bloom (in terms of presence of

microcystin-LR, -RR and -YR) in the pond. The simultaneous,

monitoring of selected ecological parameters (temperature of

water, irradiance, rainfall, pH, N and P) was also done to

decipher the possible regulation of the bloom growth and its

toxicity.

2. Materials and methods

2.1. Microcystis strain and its cultivation

Axenic culture ofM. aeruginosa NIES 843 strain (courtesy, Hee-

Mock Oh, KRIBB, Korea) was maintained in BG-11 medium

(Rippka et al., 1979). The template DNA was used as positive

control in PCR and for making plasmid and genomic DNA

standards in real-time PCR (ref. Section 2.5.1).

2.2. Sample collection

Durgakund Pond, Varanasi, India (25�1702000 N, 82�5905800 E) lies8.77 m above the sea level. The pond has an area of 8010 m2

with a mean depth 26.6 m. The pond is not connected to any

river with exception of incoming water from adjacent

temples. Every two weeks from May 2010 to April 2011, water

samples (1 L) were collected in 2 L acid washed glass bottles

from surface above a depth of 20 cm after somemixing at four

different sites in the pond. These samples were examined

microscopically and maintained at 4 �C. The samples were

pooled together and processed on the same day of collection.

For DNA extraction, the sampleswere filtered on the same day

of collection using 0.2 mm cellulose nitrate filter (Sartorius,

Germany), and stored until use for molecular analyses.

2.3. DNA extraction, PCR and sequencing

DNA was extracted from the filter by grinding in liquid

nitrogen and suspending in TE buffer (pH 8.0, 10 mM TriseHCl

and 1 mM EDTA) followed by phenol/chloroform method

(Sambrook and Russell, 2001). Primer pairs for amplification of

Microcystis sp. specific cpcBA-IGS region (Kim et al., 2010) and

mcyA condensation domain (Hisbergues et al., 2003) respon-

sible for microcystin biosynthesis were used. The reaction

was performed in a final volume of 50 mL with 5 mL of 10�buffer, 5 mL of 2.5 mM dNTP mixture, 1.5 mL of the respective

primer sets (10 pmol), 1 mL of template DNA and 5 U of Ex-Taq

DNA polymerase (Takara, Japan). The PCR protocol for cpcBA-

IGS consisted of initial denaturation at 94 �C (5 min); 30 cycles

of 94 �C (30 s), 55 �C (30 s), 72 �C (1 min) with a final extension

of 72 �C (7 min). The amplification conditions for mcyA

condensation domain consisted of pre-incubation at 94 �C(4 min); 30 cycles of 94 �C (15 s), 53 �C (15 s), 72 �C (30 s) with

a final extension step of 72 �C (7 min). PCR was performed

using a GeneAmp PCR System 2700, thermal cycler (Applied

Biosystems, Foster City, CA), and amplified products were

visualized on 1.5% agarose gels stained with ethidium

bromide under UV light. The PCR products were purified using

a Qiagen QIAquick PCR purification column and T-clonedwith

pDrive vector (Qiagen, Germany). The purified products were

sequenced using the SP6 and T7 primers by the chain-

termination method on an ABI377 automated sequencer

(Solgent Ltd., Daejeon, Korea). A total of seven plasmids were

sequenced for each sample.

2.4. Selected environmental parameters andmicrocystins determination

The water temperature and pH were measured by a m pH

system 361 (Systronics, India). The rainfall and daily light

intensity data were obtained from the meteorological section

of Department of Geophysics and Institute of Agricultural

Sciences, Banaras Hindu University, respectively. The total

nitrogen (TN) and total phosphorus (TP)were determined after

persulfate oxidation to nitrate (D’Elia et al., 1977) and ortho-

phosphate (Menzel and Corwin, 1965), respectively. Finally,

nitrate and orthophosphate were determined using a second-

derivative method (Crumpton et al., 1992) for former and an

ascorbic acid method (Eaton et al., 1995) for the latter. Total

dissolved nitrogen (TDN) and total dissolved phosphorus

(TDP) weremeasured after filtration of water samples through

0.45 mmfilters (Sartorius, Germany). A known volume of water

samples from the pond was filtered (GF/C, Whatman, UK) and

Table 1 e Validated parameters of LCeMS method forestimation of microcystin-LR, -RR and -YR.

Microcystin-LR

Microcystin-RR

Microcystin-YR

Accuracy

(%)

86e104 88e105 96e114

Precision

(% CV)

�6 �3 �5

Sensitivity

(ng/mL)

0.98 0.98 1.95

Concentration

range for

calibration

curve

(ng/mL)

3.19e500 3.19e500 3.19e500

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7 819

suspended in 80% acetone for overnight in dark (4 �C). Thesupernatant was used to measure chlorophyll a (665 nm) and

carotenoids (460 nm) according to Myers and Kratz (1955). The

residue was extracted in deionized water for phycocyanin,

and measured according to Brody and Brody (1961).

2.4.1. Microcystin extractionA known volume of water sample, depending on the

concentration of cyanobacterial cells, was filtered using a GF/

C filter (Whatman, UK). Filters containing algal cells were

lyophilized and stored until use for microcystin analysis. The

lyophilized cells were extracted three times with 20 mL of

aqueous methanol (75%) for 1 h (Fastner et al., 1998). The

extracts were rotary evaporated and dried in vacuo at 45 �C.The residue was resuspended in 2� 500 mL of methanol and

filtered through 0.20 mmfilter (Sartorius, Germany) prior to the

analysis of intracellular microcystins (MC). The filtrate was

used for determination of dissolved (extracellular) micro-

cystins using LCeMS.

2.4.2. Estimation of microcystin2.4.2.1. Microcystin standards and internal standards. Micro-

cystin-LR, -RR and -YR standards were obtained from Alexis

Biochemicals (Lausen, Switzerland). Norepinephrine was

purchased from SigmaeAldrich (St. Louis, MO, USA). Mass

grade formic acid, acetonitrile and ammonium acetate were

purchased from Merck, Germany. A stock solution of MC-LR

(5 mg/mL), MC-RR (5 mg/mL) and MC-YR (2 mg/mL) was

prepared in methanol separately. Finally, a mixture of MC-LR,

-RR and -YR (total concentration of 1000 ng/mL) working

solution was prepared in methanol. Further, it was serially

diluted in methanol up to 0.12 ng/mL. Stock solution (1 mg/

mL) of internal standard (IS) was prepared by dissolving

norepinephrine in methanol. It was further diluted in the

extraction solvent containing 70% methanol and 0.1% formic

acid to reach 500 ng/mL.

2.4.2.2. LCeMS conditions. Chromatographic separation was

achieved using Thermo Accela UHPLC system (Thermo Elec-

tron Corp, Waltham, MA, USA) with a quaternary pump con-

nected to an online degasser, autosampler and photodiode

array detector (PDA). Chromquest Software (version 4.1) was

used to control all parameters of UHPLC. Analytical separation

of the microcystin compounds was achieved on a Purospher

STAR RP-18 endcapped (3 mm particles, 55� 4 mm size)

column (Merck Darmstadt, Germany) and the column

chamber maintained at 40 �C. The mobile phase consisted of

acetonitrile containing 0.1% formic acid (A) and water with

ammonium acetate (5 mM) containing 0.1% formic acid (B)

with the linear gradient program: 0 min, 100% B; 3 min, 42% B

and 5 min, 100% B. Mobile phase was pumped at the rate of

0.5 mL/min. The autosampler tray was kept at ambient

temperature. Twenty microliter of each sample was injected

into the UHPLC with a run time of 5 min.

Mass spectrometric detection of analyte and IS was carried

out on Linear Ion Trap Quadrupole LCeMS/MS Mass Spec-

trometer 4000 Q TRAP AB Sciex instrument (ABS, Foster City,

CA, USA) equipped with a TurboIonSpray (ESI) source oper-

ated in the positive ion mode. Following MS parameters were

set: Curtain gas 20; IonSpray voltage 5.5 kV; Ion source

temperature 400 �C; Ion source gas-1 40% and Ion source gas-2

60%. The Differential Potential (DP) was set at 80 for MC-LR

and -YR and 70 for MC-RR. The Entrance Potential (EP) was

set at 10 for all the microcystins. The DP and EP for IS were set

at 31 and 6, respectively. Quantification was performed using

Single Ion Monitoring (SIM) mode based on molecular adduct

ion for MC-LR m/z 995.5; MC-RR m/z 519.79; MC-YR m/z

1045.53. The transition for norepinephrinewasm/z 170.1. Data

acquisition and integration was performed by Analyst 1.4.2.

Software (ABS, Foster City CA, USA).

2.4.2.3. Method validation and calibration curve. LCeMS

method for the estimation of MC-LR, -RR and -YR was used as

described by Spoof et al. (2003) with minor modifications,

revalidated for accuracy, precision, sensitivity and specificity.

No interfering peaks were found corresponding to the reten-

tion time of microcystin. All of the validated parameters of

this method are mentioned in Table 1. A linear calibration

curve for microcystin standards was derived from the peak to

area ratio against IS by using 3.91, 7.81, 31.3, 125 and 500 ng/

mL concentrations. Concentration of microcystin in samples

was calculated from the area ratios using the calibration

curve. The linearity of the calibration curve was also calcu-

lated, and a correlation coefficient (r2) of 0.99 or better was

selected for each microcystin variant calibration curve. The r2

for MC-LR, -RR and -YR was 0.9998, 0.9999, and 0.9997,

respectively. Lower limit of quantification was defined as the

lowest concentration with a coefficient of variance (% CV)<

20%.

2.5. Quantitative real-time PCR

DNA from samples collected between July and October were

subjected to real-time PCR analysis to quantify the gene copy

numbers of Microcystis sp. specific cpcBA-IGS and potentially

toxigenic cyanobacteria (Anabaena,Microcystis and Planktothrix

sp.)-specific mcyA (condensation domain) genes. The real-

time PCR was performed in 20 mL (final volume) of reaction

mixture containing 10 mL of iTaq SYBR Green Supermix with

ROX (Bio-Rad, Hercules, CA), 1 mL of each primer set for cpcBA-

IGS (Kim et al., 2010) and mcyA condensation domain

(Hisbergues et al., 2003) and 1 mL of template DNA using CFX 96

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7820

C 1000� Thermal cycler (Bio-Rad, Hercules, CA). The qPCR

programs consisted of an initial pre-heating step of 5 min at

94 �C; 45 cycles of 94 �C (30 s), 62 �C (cpcBA-IGS) or 60 �C (mcyA

condensation domain) for 30 s and 72 �C (30 s). All of the

samples were amplified in triplicate. Melting curve analysis of

all the samples was done by raising the temperature from

59 �C to 70 �C.

2.5.1. Standards for real-time PCRThe target genes, cpcBA-IGS and mcyA condensation domain,

were cloned into StrataClone pSC-A-amp/kan vector (Stra-

tagene, USA). Plasmid DNA was extracted using AxyPrep�Plasmid Miniprep Kit (Axygen Inc., CA, USA) according to the

manufacturer’s instructions. Inserts in the clones were

confirmed by colony PCR using primers for cpcBA-IGS (Kim

et al., 2010) and mcyA condensation domain (Hisbergues

et al., 2003). Individual standard curves were established for

the cpcBA-IGS and mcyA condensation domain using 10-fold

serial dilutions of single copy plasmid and genomic DNA from

M. aeruginosaNIES 843. Genome size of 5.8 Mb forM. aeruginosa

NIES 843 was used in the mcyA copy number calculation. The

DNA copy numbers were calculated assuming one copy each

of mcyA and cpcBA-IGS per genome as described by Vaitomaa

et al. (2003). The Ct values were automatically determined

using CFX Manager Software (version 1.5). The copy numbers

of the environmental samples were calculated using the

regression equations of the plasmid and genomic DNA stan-

dards. DNA corresponding to one cell inM. aeruginosaNIES 843

sample was calculated, and the cell abundance estimated

based on the volume of samples collected (Rinta-Kanto et al.,

2005; Baxa et al., 2010).

2.6. Statistical analysis

All experiments were carried out in triplicate with standard

deviation (SD) represented as bars wherever necessary using

Microcal� Origin� Version 6.0. Normal distribution of each

parameter was tested using ShapiroeWilk normality test,

before correlation analysis. The data distributions of micro-

cystins, TN, TDN, TPN, TDP, rainfall, phycocyanin, and chlo-

rophyll awere positively skewed, i.e. they had long upper tails.

Therefore, they were log-transformed before modeling to give

these an approximate normal distribution.

Fig. 1 e Agarose gel showing amplification products of cpcBA-IG

from the same samples of Durgakund pond (July 2010) having

template DNA; Lanes 1 and 6), positive control (template DNA f

3. Results

3.1. Identification and toxicity analysis of bloomsamples

The microscopic examination of pond samples during study

showed presence of colonial as well as single celled forms of

Microcystis sp. however, Anabaena sp. and Planktothrix sp. were

not seen in themicroscopic field. The PCR amplified product of

Microcystis sp. specific cpcBA-IGS gene (301 bp) and mcyA

condensation domain (300 bp) representing potentially toxi-

genic cyanobacterial genera (Microcystis sp., Anabaena sp., and

Planktothrix sp.) along with negative (without template DNA)

and positive (template DNA from M. aeruginosa NIES 843)

controls is represented in Fig. 1. Sequencing of purified PCR

product of cpcBA-IGS confirmed the presence of Microcystis sp.

while, sequencing of purified PCR product of mcyA conden-

sation domain and its alignment analysis ascertained the

potentially toxigenic Microcystis sp. with more than 99%

similarity to M. aeruginosa.

3.2. Microcystins, pigments and environmentalparameters (water temperature, irradiance, pH, rainfall andnutrients) in the target pond

All the three microcystins (MC-LR, -RR and -YR) were detected

in the bloom samples wherein MC-RR was predominant.

Extracellular (dissolved) and intracellular (cell bound) micro-

cystins were referred to as total microcystin for each variant

(Fig. 2). Samples spiked with standards andmicrocystins were

identified on the basis of both m/z match and retention time.

Of the total microcystin pool, MC-RR represented 49.85%

(291 mg/L) followed by MC-LR 40.57% (237 mg/L) and MC-YR

with 9.58% (56 mg/L). The levels of pigments chlorophyll a,

phycocyanin and carotenoidswere highest in early September

as 2.20, 31.29 and 1.31 mg/L, respectively (Fig. 3). Thus highest

levels of pigments and total microcystins were recorded only

in early September. Precipitation in Durgakund for one year

(May 2010 to April 2011) is represented in Fig. 4. Most of the

precipitation was in July, August and September. Highest

average rainfall (10.78 mm)was on 16 September 2010. The pH

of water samples ranged from 7.39 to 8.31 as it started to

increase in late June with the highest in early September

S (Lanes 2e4) and mcyA (condensation domain) (Lanes 7e9)

100 bp DNA marker (Lane M), negative control (without

rom Microcystis aeruginosa NIES 843; Lanes 5 and 10).

Fig. 2 e Seasonal variation in microcystin-LR, -RR and -YR

of phytoplankton in Durgakund pond as determined by

LCeMS method. The error bars represent standard

deviation values obtained in the triplicate samples.

Fig. 4 e Seasonal variation in annual rainfall for

2010e2011. The rainfall is the average value of the

previous 14 days.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7 821

(Fig. 5). Lowest water temperature (8.6 �C) was recorded in

January 2011 while the highest (37.1 �C) in April 2011. The

irradiance showed little variation and ranged from 54.00 to

82.10 MJ/m2/day (Fig. 5). TN concentration increased in late

July and attained its peak (32.8 mg/L) in August (Fig. 6). The

highest concentration (0.82 mg/L) of TP was in early

September 2010 and late February 2011 (0.90 mg/L). However,

its concentrations ranged from 0.06 mg/L to 0.11 mg/L from

May to early June (Fig. 7). TPN and TPP showed similar trends

with TN and TP, respectively. There were fluctuations in dis-

solved N and P with their highest levels in late July (5.20 mg/L)

and late February (0.05 mg/L), respectively.

3.3. Standard curves using real-time PCR and detectionrange

A standard curve developed using five dilutions of plasmid and

genomic DNA from axenic strain of M. aeruginosa NIES 843

Fig. 3 e Seasonal variation in chlorophyll a, phycocyanin,

carotenoids of phytoplankton in Durgakund pond. The

error bars represent standard deviation values obtained in

the triplicate samples.

rangedfrom1.98� 105 to1.98� 109mcyAcopiesand1.97� 105 to

1.97� 109 cpcBA-IGS copies for the plasmid DNA. The efficien-

cies were also calculated for each standard (Table 2). BothmcyA

condensation domain and cpcBA-IGS copies ranged from

1.5� 102 to 1.5� 106 for genomic DNA in a reaction. DNA

concentrations for the cpcBA-IGS standard ranged from9� 10�4

to9.3 ngofplasmidDNAand1.3� 10�3 to8.1 ngofgenomicDNA

per reaction. DNA concentrations for the mcyA condensation

domain standard ranged from 1.5� 10�3 to 8.2 ng of plasmid

DNA and 8� 10�4 to 8.1 ng of genomic DNA per reaction. The

lowest limit corresponded approximately to 155 copies/mL.

3.4. Gene copy numbers, cell abundance, dominance ofmicrocystin-producing Microcystis strains andenvironmental parameters

The abundance of cpcBA-IGS target gene was highest in

September and decreased in October (Table 3). A similar trend

Fig. 5 e Seasonal variation in pH, water temperature and

irradiance for one year (2010e2011) in Durgakund pond.

The error bars represent standard deviation values

obtained in the triplicate samples. The daily irradiance is

the average value of the previous 14 days.

Fig. 6 e Seasonal variation in concentrations of nitrogen.

The total nitrogen (TN) consists of the dissolved form (TDN)

and particulate form (TPN). The error bars represent

standard deviation values obtained in the triplicate

samples.

Table 2 e Standard curve parameters from real-time PCRanalysis of cpcBA-IGS and mcyA condensation domain ofthe cyanobacterium Microcystis sp.

Targetgenes

Standard Efficiencya Slope y-intercept R2

cpcBA-IGS Plasmid 0.937 �3.482 37.87 0.998

cpcBA-IGS M. aeruginosa

NIES 843

genomic DNA

0.899 �3.588 44.62 0.991

mcyA Plasmid 0.901 �3.583 36.79 0.985

mcyA M. aeruginosa

NIES 843

genomic DNA

0.928 �3.507 38.67 0.995

a The amplification efficiency (e) was calculated by e¼ 10�1/S� 1,

where S is the slope.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7822

was seen in case of mcyA condensation domain copies, with

the abundance up to 2e4 orders of magnitude lower, for the

study period. The ratio of potentially toxigenic Microcystis sp.

to that of total Microcystis sp. ranged from 0% to 14% with the

highest in September. Microcystis sp. dominated in the pond

ranging from 8.13� 107 to 1.03� 109 cell equivalents/L. The

potentially toxigenic cyanobacteria varied from 9.16� 104 to

1.47� 108 cell equivalents/L. It is apparent from the data that

potentially toxigenic cyanobacteria fluctuated more over that

of total Microcystis sp. in the pond indicating variation in

proportions of toxigenic cyanobacterial genera during July

2010 to October 2010. These variations correspondedwith that

of microcystin variants in the pond (Table 3).

The correlation among toxic Microcystis sp., selected envi-

ronmental parameters and levels of total microcystin variants

for one year is represented in Table 4. Rainfall and pH showed

Fig. 7 e Seasonal variation in concentrations of

phosphorus. The total phosphorus (TP) consists of the

dissolved form (TDP) and particulate form (TPP). The error

bars represent standard deviation values obtained in the

triplicate samples.

positive correlationwithall themicrocystin variants; however,

irradiance showed negative correlations with chlorophyll

a ( p¼ 0.001, R2¼ 0.372), carotenoids ( p¼ 0.001, R2¼ 0.393) and

all microcystin variants ( p¼ 0.000, R2¼ 0.458) with MC-LR,

( p¼ 0.002, R2¼ 0.338) with MC-RR and ( p¼ 0.000, R2¼ 0.424)

with MC-YR. TN showed significant correlation with chloro-

phyll a, carotenoids and MC-YR, whereas TP promoted the

toxic mcyA condensation domain copy number and had

significant correlation with chlorophyll a and carotenoids. Of

the threemicrocystinvariants,MC-LRand -RR showedpositive

correlation with mcyA condensation domain copy numbers

( p¼ 0.045, R2¼ 0.785) and ( p¼ 0.007, R2¼ 0.935), respectively.

However, in case of MC-YR, correlation was as p¼ 0.155,

R2¼ 0.543. Positive correlationswerealso found for chlorophyll

a with MC-LR ( p¼ 0.000, R2¼ 0.437), MC-RR ( p¼ 0.008,

R2¼ 0.261) and MC-YR ( p¼ 0.000, R2¼ 0.472), and for caroten-

oids with MC-LR ( p¼ 0.000, R2¼ 0.432), MC-RR ( p¼ 0.012,

R2¼ 0.237), and MC-YR ( p¼ 0.000, R2¼ 0.448). Total micro-

cystin attained its highest level (584 mg/L) during September

corresponding with the pigments concentrations (chlorophyll

a, phycocyanin and carotenoids) in the pond thus indicating

correlation between bloom and microcystins in the pond.

4. Discussion

Blooms (50%e75%) can produce toxins, often with more than

one toxin present (WHO, 2003). Microcystins are among the

most cosmopolitan cyanobacterial toxins in the lakes and

brackish waters. There are concrete evidences regarding

presence of toxic and non-toxic strains of cyanobacterial

genera in various blooms (Vaitomaa et al., 2003; Pearson and

Neilan, 2008). The present study using the conventional and

real-time PCR characterized the real state of cyanobacterial

bloom composition along with the levels of microcystin vari-

ants in the reference pond. Earlier reports of Prakash et al.

(2009) on this pond however, lacked the molecular

approaches. Some Indian workers used conventional PCR and

morphological characteristics of Microcystis sp. without

quantifying and correlating themicrocystin variants with that

of the toxin producing genes in central India (Ghosh et al.,

2008; Sangolkar et al., 2009), while Kumar et al. (2011) re-

ported the presence of small number of non-toxic Planktothrix

Table

3ecp

cBA-IGSandmcyAco

ndensa

tiondom

ain

copynum

bers

andce

llequivalents/L

oftotalM

icrocystis

sp.a

ndtoxigenic

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-RR

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22Jul2010

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wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7 823

and Anabaenopsis sp. in addition to Microcystis sp. as the

dominant genera in the target pond.We could not observe any

other cyanobacterial genera except Microcystis sp. during

microscopic examinations. Thismay be ascribed to very small

number of Planktothrix and Anabaenopsis sp. present in the

pond. Our quantifications based on real-time PCR regarding

dominance of Microcystis sp. and the presence of potentially

toxigenic Microcystis sp. verified the presence of toxigenic

Microcystis sp. using multiplex PCR. Microcystis sp. present in

the pond was composed of colonial and single celled forms.

However, Microcystis is known to have various species and

subspecies possibly because of high degree of sequence

dissimilarity in mcy genes in them (Kim et al., 2010). Many

workers have successfully used 16S rRNA gene for the quan-

tification of total Microcystis sp. (Rinta-Kanto et al., 2005; Ha

et al., 2009; Baxa et al., 2010). Taxonomic resolution offered

by 16S rRNA genes seemed insufficient to distinguish between

the closely related organisms (El Herry et al., 2009). The

genetic variations in phycocyanin operon have enabled the

intrageneric delineation of toxic cyanobacterial strains

(Neilan, 2002). The quantification of target genes present in

the pond samples must reflect the potentially toxic and non-

toxic strains, and therefore, we used Microcystis sp. specific

primers for cpcBA-IGS (Kim et al., 2010). mcyA condensation

domain was used for quantifying potentially toxigenic cya-

nobacterial population (Planktothrix sp., Anabaena sp. and

Microcystis sp.) in the same pond, however, sequencing of PCR

products of mcyA condensation domain showed more than

99% similarity toM. aeruginosa thus confirming the dominance

of potentially toxigenic Microcystis sp. in the bloom. This

implied that the presence of higher level of microcystin

especially MC-RR along with other variants (MC-LR and MC-

YR) was only the contribution of potentially toxigenic Micro-

cystis sp. Although hepatotoxigenic Microcystis is well known

for its health risks but there are no data available on people

getting affected using pond water frequently for religious

purposes. Therefore, the identification and quantification of

toxic and non-toxic Microcystis sp. in such ponds seemed

imperative. The induction of the growth and development of

toxic Microcystis bloom is not yet known in spite of the fact

that cyanobacterial blooms are regulated by various environ-

mental factors including nutrient availability (Sivonen, 1990).

Durgakundpondwater sampleswere analyzed periodically

for pigments (chlorophyll a, phycocyanin and carotenoids)

and microcystin variants (MC-LR, -RR and -YR) for one year.

The data in Figs. 2 and 3 clearly showed the highest micro-

cystins and pigments content during July to October 2010 with

their climax in September. The concentrations of chlorophyll

a, an indicator of phytoplankton biomass, were well above the

guidance level of 10 mg/L for relatively low probability of

adverse health effects (WHO, 2003). Increase in pigments

content indicated rise in nutrient(s) level of target water body.

Detection and quantification of all the threemicrocystins (MC-

LR, -RR and -YR) in the bloom using LCeMS, confirmed their

presence in the pond water with the predominance of MC-RR

in all the samples (Fig. 2). Such observations are in tune with

those of Prakash et al. (2009). Although microcystin-LR is

mentioned as the most frequently occurring microcystin but

its co-occurrence with MC-RR and -YR is also reported (Zhang

et al., 1991; Kemp and John, 2006). MC-RR often predominates

Table 4 e Coefficient of determination (R2) between selected environmental parameters and cyanobacteria toxicity.

Environmental parameters Pigments (mg/L) Copy numbers Microcystin variants (mg/L)

Chlorophyll a Phycocyanin Carotenoids cpcBA-IGS mcyA -LR -RR -YR

1. pH 0.080 0.216* 0.077 0.003 0.133 0.194* 0.190* 0.270**

2. Water temp. 0.036 0.005 0.004 0.024 0.003 0.031 0.036 0.048

3. Rainfall 0.270** 0.002 0.255** 0.026 0.024 0.534** 0.456** 0.533**

4. Irradiancea 0.372** 0.004 0.393** 0.004 0.208 0.458** 0.338** 0.424**

5. TN 0.396** 0.075 0.296** 0.054 0.003 0.105 0.034 0.163*

6. TDN 0.373** 0.016 0.167* 0.002 0.002 0.027 0.000 0.049

7. TPN 0.274** 0.070 0.268** 0.049 0.000 0.093 0.035 0.147

8. TP 0.643** 0.084 0.438** 0.402 0.818* 0.069 0.000 0.092

9. TDP 0.092 0.123 0.077 0.365 0.665 0.000 0.005 0.004

10. TPP 0.658** 0.072 0.662** 0.400 0.819* 0.076 0.001 0.097

Where 2: water temperature (�C), 4: irradiance (MJ/m2/day), 5: total nitrogen, 6: total dissolved nitrogen, 7: total particulate nitrogen, 8: total

phosphorus, 9: total dissolved phosphorus and 10: total particulate phosphorus, *p< 0.05, **p< 0.01.

a Irradiance showed significant negative correlation.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7824

in warmer climates as supported by the laboratory experi-

ments of Rapala et al. (1997) where Anabaena strains produced

high levels of MC-LR and MC-RR at lower and higher temper-

atures, respectively. Van de Waal et al. (2010) reported

enhanced nitrogen availability to increase the intracellular

content of the nitrogen-rich amino acid arginine, and thereby

promoting production of the [Asp3] microcystin-RR variant.

Thus, the dominance of MC-RR in the pond samples may be

explained on this ground. The differing levels of microcystin

variants in pond necessitate considering all microcystin vari-

ants to make water quality index in the Indian context.

An and Jones (2000) are of the opinion that an intense

monsoon reduces while the weak one increases the cyano-

bacterial bloom in Korea. The pigment concentrations

increased with rainfall on 22 July 2010 and then decreased

with the next heavy rainfall on 16 September 2010 thus indi-

cating decrease in the cyanobacterial population (Figs. 3 and

4). This is in accordance with the report of Ahn et al. (2002).

Rainfall showed positive correlation with the microcystin

variants, chlorophyll a and carotenoids. The highest pH was

recorded at the time of bloompeak (Fig. 5) and it had a positive

correlation with the phycocyanin and all the three micro-

cystin variants (Table 4). Low temperature and acidic pH

supported growth of eukaryotic algae (Paerl et al., 2001). High

water temperature supported cyanobacterial blooms

throughout the year but could not be correlated with micro-

cystins and pigments (Table 4). The irradiance was low

(54.21 MJ/m2/day) during the bloom peak during early

September (Fig. 5). In this study, irradiance had a negative

correlation with chlorophyll a, carotenoids and all three

variants of microcystin and could possibly reduce the func-

tionality of reaction centers in surface blooms. Cyanobacterial

growth depends upon the irradiance as inverse correlation

was observed between pigment content and irradiance

(Tandeau de Marsac and Houmard, 1993). Extremes of pH

(acidic or alkaline) render most of the nutrients unavailable to

the organisms. Therefore, the total particulate and dissolved

nitrogen and phosphorusmay be regulatedwith varying pH in

the pond. TN level was maximum in September when cya-

nobacterial biomass was at its climax (Fig. 6). There was

significant correlation of chlorophyll a, carotenoid and MC-YR

with nitrogen but it is difficult to evaluate the role of nitrogen

in microcystin production (Table 4). Ha et al. (2009) also re-

ported similar positive correlations with that of chlorophyll

a and microcystin. However, Hotto et al. (2008) reported

aweak correlation of chlorophyll awith nitrate and absence of

heterocysts in Anabaena and Aphanizomenon sp. suggesting

that Oneida Lake was not nitrogen limited.

As far as P was concerned, TP was maximum in September

2010 (0.82 mg/L) and February 2011 (0.90 mg/L) thus indicating

that cells could have accumulated enough P (Fig. 7). However,

it is very difficult to comment on the role of P in microcystin

production by M. aeruginosa. There are differing views in this

regard. Davis et al. (2009) observed that P never affected

Microcystis growth in eutrophic systems. Vezie et al. (2002)

found that at higher P concentrations, growth rate of toxic

Microcystis sp. exceeded the non-toxic strains. There are

reports that P increased microcystin levels (Lee et al., 2000;

Chorus et al., 2001; Gupta et al., 2001). The increased copy

numbers of mcyA condensation domain indicated the pres-

ence of increased number of toxic strains during September

2010 thereby increasing the microcystin concentration (Table

3), however, P levels could not be correlated with the micro-

cystin variants (Table 4). This is in accordance with the find-

ings of Ha et al. (2009). By contrast, Oh et al. (2000) reported

increased microcystin content under P limited conditions. In

another report, Utkilen and Gjølme (1995) observed that

nitrate and phosphate limited conditions had no effect on

toxin production byM. aeruginosa. Tilman et al. (1982) reported

80e90% of cyanobacterial population at a TN:TP ratio of 12.

Cyanobacterial dominance at a low TN:TP ratio was also re-

ported in Daechung Reservoir (Ahn et al., 2002). TN:TP weight

ratio in Durgakund pond varied from2 to 57 but the bloomwas

at its peak at a lower ratio of 9 (Figs. 6 and 7).

Samples during July to October 2010 when the bloom

dominated were subjected to real-time PCR analysis. Real-

time PCR efficiency was calculated for the standard curves

as shown in Table 2. Microcystin-producing mcyA condensa-

tion domain copy numbers or toxigenic cyanobacterial cell

abundance were high in early September and corresponded

with the microcystins concentration (Table 3) with a strong

positive correlation ( p< 0.001, R2¼ 0.988 or p< 0.001,

R2¼ 0.986). Previous studies employing different mcy genes

also showed a positive correlation with microcystin

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 8 1 7e8 2 7 825

concentrations (Hotto et al., 2008; Ha et al., 2009). There was

a significant relationship betweenmicrocystin concentrations

and chlorophyll a ( p< 0.0001, R2¼ 0.905). This is in conformity

with the previous studies on relationships between micro-

cystin and chlorophyll a concentration (Kotak et al., 2000;

Chorus et al., 2001; Ha et al., 2009). The microcystin concen-

tration can be effectively correlated with mcyA condensation

domain gene copy numbers, and real-time PCR could be used

for rapid approximate estimation of abundance of toxigenic

cyanobacterial genera in pond waters.

Potentially toxigenic Microcystis cell abundance was up to

2e4 orders lower than the total Microcystis sp. over the entire

study period (Table 3). There was an increase in cpcBA-IGS copy

numbers from 22 July to 5 August 2010 with the maximum

recorded in September with the blooms on their climax. A

sudden decrease in cpcBA-IGS copy numbers on 19th August

2010 may be attributed to simultaneous run-off water from

temple to the pond thus diluting the bloom population flora,

therebyaffectingthesampling.Theratioofpotentially toxigenic

Microcystis sp. with that of the total Microcystis sp. ranged from

0% to 14% and the highest during the bloom peak. Thus it is

evident that most toxic strains appeared as conditions were

most favorable. In similar studies, the ratio varied from 0% to

11%(Doblinetal., 2007), 0%to37%(Hottoetal., 2008), 0.7%to41%

with average of 12% (Ha et al., 2009) and 0.3% to 35% (Yoshida

et al., 2007). Thus, it may be ascribed that toxic/non-toxic cya-

nobacterial strains in bloom occurs at varying environmental

conditions in different regions, and studies on quantification of

toxic and non-toxic Microcystis sp. and/or cyanobacterial sp.

seemed necessary for making the water quality index and also

in the formulations of preventive measures to avoid human

health hazards. However at present, there is a lack of data

dealing with frequent exposure of people to such ponds.

5. Conclusions

The potability of thewater is amajor global concern, and there

are stray reports on toxin assessments in water bodies in

India. From this study, it can be concluded as:

� It is very difficult to correlate water quality parameters and

microcystin concentration except the growth of cyano-

bacterial population.

� Two peaks of water quality and MCs were generally

observed in a biweekly sampling owing to the changes in

rainfall, light irradiance and temperature.

� MC-RR was the dominant variant with the concentration

beyond the set limit of WHO.

� Real-time PCR could be used successfully in the quantifica-

tion of toxic and non-toxic cyanobacteria for the first time in

the Indian water body.

Acknowledgements

We are grateful for the facilities extended to Ankita Srivastava

by Dr. Hee-Mock Oh (a grant from Advanced Biomass R&D

Center, MEST), Director of Biosystems Research Group, KRIBB,

Korea. We are also thankful to All India Institute of Medical

Sciences (AIIMS) for providing the DST Central Facility for

LCeMS. Financial support from DST (Department of Science

and Technology), New Delhi (DST/INSPIRE fellowship/2010, IF

10355, Dt. 26 November 2010) to Ankita Srivastava as JRF is

gratefully acknowledged. RKA is thankful to UGC (University

Grants Commission), New Delhi, Project Code No. P-01/623 for

financial support.

Appendix. Supplementary material

Supplementary material associated with this article can be

found, in theonlineversion, atdoi:10.1016/j.watres.2011.11.056.

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