Flow Cytometric Analysis of Freshwater Cyanobacteria: A Case Study

: Eutrophication is a process that occurs due to the excessive accumulation of nutrients, primarily nitrogen and phosphorus, from natural and anthropogenic sources. This phenomenon causes cyanobacterial overgrowth, which over time leads to cyanobacterial harmful algal blooms (CHABs) that a ﬀ ect public drinking water sources and water sites with recreational usage. The rapid detection of bloom-forming cyanobacteria in freshwater bodies is critical in order to implement prevention strategies. Cyanobacteria contain phycobiliproteins such as phycoerythrin and allophycocyanin as part of the phycobilisome that allows autoﬂuorescence. In this study, samples from 36 freshwater bodies in 14 New Jersey counties were collected and analyzed using ﬂow cytometry with forward-scatter phycoerythrin and allophycocyanin parameters. and Microcystis aeruginosa were used as references. The results revealed that 17 out of the 36 analyzed sites contained all three references and related species. Seven sites showed Microcystis and Cylindrospermum -like species, while four sites indicated Microcystis and Cylindrospermum -like species. Six water bodies showed Cylindrospermum -like species, and two sites showed Microcystis -like species. Polymerase chain reaction (PCR)-based assays further conﬁrmed the ﬂow cytometric results. The ﬁndings from this study suggest that ﬂow cytometry could potentially serve as a rapid method for freshwater cyanobacteria detection and screening.


Introduction
Cyanobacteria, also known as blue green algae, are some of the earliest dated oxygen-releasing prokaryotes, originating about 3.5 billion years ago [1]. Their size ranges from 0.5 to 50+ µm, and they may be composed of different cellular arrangements, such as unicellular Synechococcus spp., filamentous Cylindrospermum spp., and colonial Microcystis spp. Cyanobacteria can be found in marine and freshwater environments, including lakes, ponds, rivers, brackish water and water reservoirs. Moreover, these bacteria can be terrestrial, colonize in volcano ashes, and even colonize in the human gut. Cyanobacteria are able to survive and outcompete other biological species in eutrophied water bodies and, consequently, can over-grow nutrients such as nitrogen and phosphorus, leading to the formation of algal blooms. Climate change as well as human disruptions such as agriculture, industrial and recreational activities, and sewage effluent all lead to eutrophication [2]. The bloom masses may cover the water surface, lowering the dissolved oxygen and causing hypoxia in the water. Although not all cyanobacteria are toxic, many possess the ability to release toxins and produce secondary metabolites known as cyanotoxins, including hepatotoxins, neurotoxins and dermatoxins, which are toxic to other cells, organisms, fish, animals and humans. Cyanobacterial harmful algal blooms (CHABs) are considered a significant concern to the ecosystem. In addition, their toxins can be very dangerous to the public, who utilize the water for drinking and recreational purposes. Cyanobacterial toxicity can vary from one bloom to another, where specific species can secrete a certain toxin or even

Water Sample Collection and Processing
Water samples from 36 water bodies (250 mL per site in Nalgene bottles) in New Jersey ( Figure 1) were obtained by the New Jersey Department of Environmental Protection (NJDEP) in summer (May to August), 2015. These water bodies are utilized for recreational purposes, drinking water, or water supply reservoirs. The Global Positioning System (GPS) coordinates as well as water temperature ( • C), pH and dissolved oxygen (mg/L) were measured and recorded on site. Water samples were collected one meter below the surface.

Water Sample Collection and Processing
Water samples from 36 water bodies (250 mL per site in Nalgene bottles) in New Jersey ( Figure  1) were obtained by the New Jersey Department of Environmental Protection (NJDEP) in summer (May to August), 2015. These water bodies are utilized for recreational purposes, drinking water, or water supply reservoirs. The Global Positioning System (GPS) coordinates as well as water temperature (°C), pH and dissolved oxygen (mg/L) were measured and recorded on site. Water samples were collected one meter below the surface.

Filtration
A Thermo Scientific™ Nalgene™ (Thermo Scientific, Rochester, NY, USA) vacuum filtration unit with polycarbonate membrane filters (Sterlitech Corporation, Kent, WA, USA) was used for filtration. The water samples were thoroughly mixed and serial filtered through 30 and 0.4 µm membrane filters, sequentially.

Flow Cytometry
Flow cytometric assays were conducted using a MACSQuant ® Analyzer, calibrated with MacsQuant calibration beads (Miltenyi Biotec, Inc., SanDiego, CA, USA). The three standard reference cultures Microcystis, S. IU 625 and Cylindrospermum spp. with Optical Density (OD750nm) of 0.591, 0.521 and 0.640, respectively, were run through the flow cytometer at fixed voltage settings with various dilutions. The water samples for each site and the <30 µm filtrates were run through the flow cytometer, with the same voltage settings as the standards. The low flow rate and mix sample options were selected for this analysis. The voltage channels for forward scatter (FSC), side scatter (SSC), B2 phycoerythrin (PE), R1 allophycocyanin (APC) were 250 V, 200 V, 800 V, and 275 V, respectively. Each uptake contained 100 μL of the sample. The analysis was carried out using FSC and PE gating strategy with the software FlowJo vX.0.7 (Tree Star, Inc., Ashland, OR).

Filtration
A Thermo Scientific™ Nalgene™ (Thermo Scientific, Rochester, NY, USA) vacuum filtration unit with polycarbonate membrane filters (Sterlitech Corporation, Kent, WA, USA) was used for filtration. The water samples were thoroughly mixed and serial filtered through 30 and 0.4 µm membrane filters, sequentially.

Flow Cytometry
Flow cytometric assays were conducted using a MACSQuant ® Analyzer, calibrated with MacsQuant calibration beads (Miltenyi Biotec, Inc., SanDiego, CA, USA). The three standard reference cultures Microcystis, S. IU 625 and Cylindrospermum spp. with Optical Density (OD 750nm ) of 0.591, 0.521 and 0.640, respectively, were run through the flow cytometer at fixed voltage settings with various dilutions. The water samples for each site and the <30 µm filtrates were run through the flow cytometer, with the same voltage settings as the standards. The low flow rate and mix sample options were selected for this analysis. The voltage channels for forward scatter (FSC), side scatter (SSC), B2 phycoerythrin (PE), R1 allophycocyanin (APC) were 250 V, 200 V, 800 V, and 275 V, respectively. Each uptake contained 100 µL of the sample. The analysis was carried out using FSC and PE gating strategy with the software FlowJo vX.0.7 (Tree Star, Inc., Ashland, OR, USA).

Molecular Assays
Chelex DNA extraction was conducted for all collected water samples. Polymerase chain reaction (PCR) was carried out with CYA359F/CYA781R, the primer set that amplifies cyanobacterial 16S rRNA genes [39], to confirm the flow cytometry results. MSF/MSR, a primer set to detect the presence of Microcystin Synthetase A Gene (mcyA) in the Cyanobacterial Genus Microcystis was also included [40]. A 1% agarose gel electrophoresis was used to visualize the PCR products. PCR products were subsequently sequenced with an ABI PRISM ® 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Multiple Sequence Comparison by Log-Expectation (MUSCLE) 3.8 was utilized to analyze the 16S rRNA genes of Cylindrospermum spp., Microcystis spp., and Synechococcus spp. [41].

Water Collection Sites
Water properties were recorded for all 36 sites (Table 1). Among all the water samples collected and tested, the water temperature ranged from 16.97 (NJLM 0213) to 30.9 • C (Alloway Lake). The pH ranged from 4.09 (Mt. Misery Lake) to 9.75 (Cooper Lake), with the majority of sites between 6 and 9. The dissolved oxygen ranged from 1.66 (NJLM 0213) to 19.1 mg/L (Cooper Lake).

Filtration Cytometric Analysis of Three Reference Cyanobacteria
Three reference species, namely, Synechococcus sp. IU 625, Microcystis aeruginosa, and Cylindrospermum spp., were measured and analyzed through flow cytometry. The results revealed that M. aeruginosa is detectable at an accurate range from an OD 750nm of 0.001 and 4.196 × 10 3 cells/mL up to an OD 750nm of 0.591 and 2.0 × 10 6 cells/mL, based on the phycoerythrin (PE) and allophycocyanin (APC) parameters ( Figure 2). Synechococcus sp. IU 625 can be accurately detected at an OD 750nm range of 0.008 to 0.521 and cell count of 2.787 × 10 3 to 1.878 × 10 5 cells/mL ( Figure 2). The detectable range of Cylindrospermum spp. spans from an OD 750nm of 0.001 to 0.640 and cell count of 1.479 × 10 3 to 7.21 × 10 5 cells/mL ( Figure 2). M. aeruginosa contains higher PE and APC intensities when compared with S. IU 625 and Cylindrospermum spp.

Flow Cytometric Analysis of 36 Water Bodies in New Jersey
Samples from a total of 36 water bodies across NJ were collected and analyzed using flow cytometric assays. Three pure cyanobacterial strains, Cylindrospermum spp., Microcystis aeruginosa and Synechococcus sp. IU 625, were included in this analysis. Using a color-gating algorithm with the forward-scatter (FSC) versus phycoerythrin (PE) parameters, the reference cyanobacteria were shown in three populations: Cylindrospermum spp. in red, Microcystis aeruginosa in orange, and Synechococcus sp. IU 625 in blue color (Figure 3 Flow cytometric analyses showed that 17 water bodies, including Deverson Lake, Lake Pachoango, Lake Pemberton, Cooper Lake, Hands Mill, Lake Morski Oko, Alloway Lake, Parvin

Flow Cytometric Analysis of 36 Water Bodies in New Jersey
Samples from a total of 36 water bodies across NJ were collected and analyzed using flow cytometric assays. Three pure cyanobacterial strains, Cylindrospermum spp., Microcystis aeruginosa and Synechococcus sp. IU 625, were included in this analysis. Using a color-gating algorithm with the forward-scatter (FSC) versus phycoerythrin (PE) parameters, the reference cyanobacteria were shown in three populations: Cylindrospermum spp. in red, Microcystis aeruginosa in orange, and Synechococcus sp. IU 625 in blue color (Figure 3).

Microcystis spp. and Synechococcus sp. IU 625 Related Species
For seven out of the 36 water bodies, including NJLM 1286 (Shadow Lake), Mirror Lake, NJLM 0315, Iona Lake, NJLM 1045 (Cifrese Lake), Laidlaw Pond, and NJLM 0754, it was revealed that the phytoplanktons in these sites contained cyanobacteria of mostly Microcystis spp. (orange) and Synechococcus sp. IU 625 (blue) related species, based on the FSC vs. PE profiles ( Figure 3B).

Microcystis spp, and Cylindrospermum spp. Related Species
For four out of the 36 water bodies, including Atlantic City Reservoir, McCarthys Lake, Amwell Lake, and Mashipacong Pond, it was shown that the phytoplankton in these sites contained mostly Microcystis spp. (orange) and Cylindrospermum spp. (red) related species, based on the FSC vs. PE profiles ( Figure 3C).

Cylindrospermum spp. Related Species
Six out of the 36 water bodies, including NJLM 0489, Roosevelt Lake, and Hunts Pond, revealed primarily Cylindrospermum spp. (red) related species ( Figure 3D). The results also showed that the cyanobacteria in NJLM 1034, Prospertown Lake, and Green Turtle Lake water bodies contained mainly Cylindrospermum spp. (red) related species and an undetermined population, based on the FSC vs. PE profiles ( Figure 3D). Flow cytometric analyses showed that 17 water bodies, including Deverson Lake, Lake Pachoango, Lake Pemberton, Cooper Lake, Hands Mill, Lake Morski Oko, Alloway Lake, Parvin Lake, Rainbow Lake, Union Lake, Great Gorge Lake, NJLM 0213, NJLM 0378, Silver Lake, Upper East Highland, Watchu Pond, and Deer Park Pond, contained cyanobacteria including Microcystis spp. (orange), Synechococcus spp. IU 625 (blue), Cylindrospermum spp. (red), and their related species based on the FSC vs. PE profiles ( Figure 3A).

Microcystis spp. and Synechococcus sp. IU 625 Related Species
For seven out of the 36 water bodies, including NJLM 1286 (Shadow Lake), Mirror Lake, NJLM 0315, Iona Lake, NJLM 1045 (Cifrese Lake), Laidlaw Pond, and NJLM 0754, it was revealed that the phytoplanktons in these sites contained cyanobacteria of mostly Microcystis spp. (orange) and Synechococcus sp. IU 625 (blue) related species, based on the FSC vs. PE profiles ( Figure 3B).

Microcystis spp., and Cylindrospermum spp. Related Species
For four out of the 36 water bodies, including Atlantic City Reservoir, McCarthys Lake, Amwell Lake, and Mashipacong Pond, it was shown that the phytoplankton in these sites contained mostly Microcystis spp. (orange) and Cylindrospermum spp. (red) related species, based on the FSC vs. PE profiles ( Figure 3C).

Cylindrospermum spp. Related Species
Six out of the 36 water bodies, including NJLM 0489, Roosevelt Lake, and Hunts Pond, revealed primarily Cylindrospermum spp. (red) related species ( Figure 3D). The results also showed that the cyanobacteria in NJLM 1034, Prospertown Lake, and Green Turtle Lake water bodies contained mainly Cylindrospermum spp. (red) related species and an undetermined population, based on the FSC vs. PE profiles ( Figure 3D).

Microcystis spp. and Related Species
The results showed that Mt. Misery and Franklinville Lake contained cyanobacteria of mostly Microcystis spp. (orange) related species, based on the FSC vs. PE profiles ( Figure 3E). Franklinville Lake water samples showed another phycoerythrin-rich population.

Molecular Analysis of Cyanobacteria
Cyanobacteria were detected in all 36 sites (data not shown) [42]. The PCR products were sequenced, and the multiple sequence alignment of the 16S rRNA from the three reference cyanobacterial strains, Cylindrospermum spp., Microcystis aeruginosa and Synechococcus spp., is shown in Figure 4. The results showed that Mt. Misery and Franklinville Lake contained cyanobacteria of mostly Microcystis spp. (orange) related species, based on the FSC vs. PE profiles ( Figure 3E). Franklinville Lake water samples showed another phycoerythrin-rich population.

Molecular Analysis of Cyanobacteria
Cyanobacteria were detected in all 36 sites (data not shown) [42]. The PCR products were sequenced, and the multiple sequence alignment of the 16S rRNA from the three reference cyanobacterial strains, Cylindrospermum spp., Microcystis aeruginosa and Synechococcus spp., is shown in Figure 4.

Discussion
In this study, water samples from 36 water bodies throughout 14 New Jersey counties were analyzed via flow cytometry. The water chemistry, including water temperature (°C), pH, and dissolved oxygen, was recorded by NJDEP, as seen in Table 1. Among all sites, NJLM 0213 contained the lowest water temperature and dissolved oxygen, with 16.97 °C and 1.66 mg/L, respectively. The highest water temperature was 30.9 °C from Alloway Lake. The highest pH and dissolved oxygen was found in Cooper Lake, with 9.75 and 19.1 mg/L, respectively. The lowest pH was 4.09 from Mt. Misery Lake. The optimal pH range for cyanobacteria is between 6 and 9 [4]. Most of the sites analyzed fall within that range, allowing cyanobacteria to grow and possibly accumulate (Table 1). Dissolved oxygen is a water property that is crucial where there are concentrations of less than 4 mg/L, which can be considered hypoxic to the water ecosystem [43,44]. Four sites from Sussex County, NJLM 0213, NJLM 0378, Silver Lake and Watchu Pond, contained very little dissolved oxygen, with 1.66, 2.65, 3.32 and 3.69 mg/L, respectively (Table 1), and showed the presence of all three reference Microcystis spp., Synechococcus sp. IU 625, Cylindrospermum spp. and related species as expected ( Figures 3A). All three cyanobacterial reference species were found in wide ranges of temperature (16.97 °C to 30.9 °C), pH (4.44 to 9.75) and dissolved oxygen (1.66 to 19.17 mg/L). Microcystis spp. was detected in the environment with pH as low as 4.09.
The water profile of each of the 36 New Jersey water sites was obtained via flow cytometric analysis in about 4 to 5 min, per sample. Thus, this study further confirmed that flow cytometry can be used to rapidly process, screen and accurately detect a large volume of environmental water samples. The profile of different cyanobacteria species can be directly detected in real time without staining or lysis, due to cyanobacterial autofluorescence. Thus, flow cytometry allows early preventive measurements to be implemented and allows for a more cost-and time-efficient routine

Discussion
In this study, water samples from 36 water bodies throughout 14 New Jersey counties were analyzed via flow cytometry. The water chemistry, including water temperature ( • C), pH, and dissolved oxygen, was recorded by NJDEP, as seen in Table 1. Among all sites, NJLM 0213 contained the lowest water temperature and dissolved oxygen, with 16.97 • C and 1.66 mg/L, respectively. The highest water temperature was 30.9 • C from Alloway Lake. The highest pH and dissolved oxygen was found in Cooper Lake, with 9.75 and 19.1 mg/L, respectively. The lowest pH was 4.09 from Mt. Misery Lake. The optimal pH range for cyanobacteria is between 6 and 9 [4]. Most of the sites analyzed fall within that range, allowing cyanobacteria to grow and possibly accumulate (Table 1). Dissolved oxygen is a water property that is crucial where there are concentrations of less than 4 mg/L, which can be considered hypoxic to the water ecosystem [43,44]. Four sites from Sussex County, NJLM 0213, NJLM 0378, Silver Lake and Watchu Pond, contained very little dissolved oxygen, with 1.66, 2.65, 3.32 and 3.69 mg/L, respectively (Table 1), and showed the presence of all three reference Microcystis spp., Synechococcus sp. IU 625, Cylindrospermum spp. and related species as expected ( Figure 3A). All three cyanobacterial reference species were found in wide ranges of temperature (16.97 • C to 30.9 • C), pH (4.44 to 9.75) and dissolved oxygen (1.66 to 19.17 mg/L). Microcystis spp. was detected in the environment with pH as low as 4.09.
The water profile of each of the 36 New Jersey water sites was obtained via flow cytometric analysis in about 4 to 5 min, per sample. Thus, this study further confirmed that flow cytometry can be used to rapidly process, screen and accurately detect a large volume of environmental water samples. The profile of different cyanobacteria species can be directly detected in real time without staining or lysis, due to cyanobacterial autofluorescence. Thus, flow cytometry allows early preventive measurements to be implemented and allows for a more cost-and time-efficient routine sampling method. As seen in Figure 3, cyanobacteria were successfully detected in all 36 collected water sites when analyzed by flow cytometry. Most of the cyanobacteria or phytoplankton species were mainly phycoerythrin-rich. It is important to note that reference cyanobacterial cultures were maintained in optimal conditions, which may cause them to exhibit different pigment levels when compared with the same species in lakes. Furthermore, PCR-based assays were conducted as a complementary method to confirm the flow cytometric analysis results [42,45]. The primers utilized were to amplify 16S rRNA genes for each reference cyanobacterium [39]. A microcystin synthetase primer was also included for cyanotoxin detection. The results from PCR-based assays indicated the presence of three reference cyanobacteria in all sites, but no microcystin was detected in any of the testing water samples.
Finally, this study highlighted the use of flow cytometry for the rapid detection of cyanobacteria species in 36 New Jersey water bodies. Flow cytometry is an efficient, rapid routine water sample testing method [46], so a larger volume sampling of freshwater bodies can be achieved. A flow cytometer with sorting capability could further isolate various phytoplankton taxa from mixed environmental samples in water bodies [47]. In addition, portable flow cytometers are available for the samples to be processed on site for more accurate analyses. In summary, flow cytometry could be used as an accompanying method for the rapid detection of cyanobacteria.