Removal of cyanobacteria from a water supply reservoir by sedimentation using flocculants and suspended solids as ballast: Case of Legedadi Reservoir (Ethiopia).

The massive growth of potentially toxic cyanobacteria in water supply reservoirs, such as Legedadi Reservoir (Ethiopia), poses a huge burden to water purification units and represents a serious threat to public health. In this study, we evaluated the efficiency of the flocculants/coagulants chitosan, Moringa oleifera seed (MOS), and poly-aluminium chloride (PAC) in settling cyanobacterial species present in the Legedadi Reservoir. We also tested whether coagulant-treated reservoir water promotes cyanobacteria growth. Our data showed that suspended solids in the turbid reservoir acted as ballast, thereby enhancing settling and hence the removal of cyanobacterial species coagulated with chitosan, Moringa oleifera seed, or their combination. Compared to other coagulants, MOS of 30 mg/L concentration, with the removal efficiency of 93.6%, was the most effective in removing cyanobacterial species without causing cell lysis. Contrary to our expectation, PAC was the least effective coagulant. Moreover, reservoir water treated with MOS alone or MOS combined with chitosan did not support any growth of cyanobacteria during the first two weeks of the experiment. Our data indicate that the efficacy of a flocculant/coagulant in the removal of cyanobacteria is influenced by the uniqueness of individual lakes/reservoirs, implying that mitigation methods should consider the unique characteristic of the lake/reservoir.

1. Introduction

Eutrophication-related deterioration of the quality of freshwaters has become an environmental issue of global concern [1] and will remain to be the most important water quality problem in the future [2]. Global climate change and anthropogenic nutrient input have resulted in frequent occurrences of cyanobacterial blooms [3,4]. Cyanobacterial bloom is a serious concern since it results in changes in the odor and taste of water. It is associated with the production of potent toxins, which present a threat to the health of humans and animals [5]. Consequently, cyanobacterial blooms may impair recreational activities, such as angling, boating, swimming, irrigation, aquaculture, and drinking water production [6].

Sixty percent of the population of Addis Ababa city, which is 2.75 Million people, depends on the Legedadi and Geffersa Reservoirs as sources of drinking water supply. However, potentially toxic and persistent cyanobacterial blooms dominated by Microcystis aeruginosa and several Anabaena spp. have recurred annually in Legedadi Reservoir [7,8]. As cyanobacterial blooms in eutrophic water bodies present a high public health risk, the immediate reduction of the potential hazard is imperative. Even though the reservoir has suffered from eutrophication-related water quality problems for decades, reports on efforts made to control the cyanobacteria using environmentally friendly restoration techniques are absent. However, the application of algaecides like copper sulfate has been practiced in the study reservoir for more than three decades. The main drawback of using copper sulfate is the lysis of cyanobacterial cells, which results in the release of toxins into the water bodies. Cell lysis caused by algaecides may lead to intoxication of aquatic organisms and eventually of humans [9].

To tackle cyanobacteria-related problems and restore water bodies, the first rational option is the reduction of external nutrient loading. However, this option is not always feasible [10], especially in developing countries like Ethiopia, where agriculture is expanding and the application of fertilizers is widely practiced. The disposal of untreated wastewater into reservoirs/lakes and the huge cost incurred by the establishment of proper wastewater treatment plants (WWTP) complicate the problem further [11,12]. Therefore, effect-oriented measures would be suitable for controlling nuisance/harmful cyanobacteria, especially for developing countries where control of external nutrient loading is not economically a preferred option at this moment. An effect-oriented technique has its own drawbacks, including its short-lived effects and the need to repeat it regularly. Thus, there is a need to look for fast, easy, cheap, and safe technologies as curative/effect-oriented methods. To this end geochemical engineering approaches, such as “floc and lock” and “floc and sink”, have been developed [13,14]. In the floc and sink technique, flocculants/coagulants such as chitosan, poly aluminium chloride (PAC), iron chloride, and Moringa oleifera seed-extract (MOS) with ballast materials (clays, natural soils, lanthanum-modified bentonite, and Phoslock) have been used to effectively coagulate cyanobacteria as intact cells and to settle to the sediment of different lakes/reservoirs [14].

The ‘floc and lock’ technique has been applied effectively in two isolated and stratifying lakes, Lake Rauwbraken [13,15] and Lake De Kuil [16] in the Netherlands, while ‘floc and sink’ has been tested effectively in an isolated bay, Lake Taihu [17] in China.

Prior to implementing such measures, in-depth research and recognition of the unique feature of the water body of concern is needed [18,19]. Those include hydro-morphological and physico-chemical features, the biology of the system, and for instance availability of local soils with natural P binding capacities [20]. In Legedadi Reservoir, the high turbidity caused by particulate materials entering the reservoir through rivers was considered a unique feature. In previous studies, ballast materials such as clay and soil were added with a coagulant to lake/reservoir water [14,20]. The high turbidity of the Legedadi Reservoir, however, was expected to bring sufficient autochthonous ballast such that, adding additional ballast is not needed. Accordingly, the floc and sink technique was implemented by directly adding only a flocculant/coagulant to the Legedadi Reservoir water.

The addition of a coagulant to reservoir water was expected to clear the water column from cyanobacteria and suspended solids. Consequently, the reservoir water was expected to become clear and light limitation would no longer prevail. As Legedadi Reservoir is a nutrient-rich reservoir, a newly cleared water column condition could allow light penetration to greater depth thereby promoting cyanobacteria growth. Therefore, an experiment was conducted to determine what could possibly happen in terms of cyanobacterial growth as the reservoir water becomes clear.

We hypothesized that PAC, MOS extract, and chitosan in a turbid water body would effectively remove cyanobacteria without the need for adding external ballast material. It was also hypothesized that, the reservoir water treated with MOS extract would lead to delayed growth of cyanobacteria due to the antibacterial effect of MOS. The laboratory experiments were conducted using reservoir samples to evaluate the efficacy of different doses of PAC, MOS extract, chitosan, and a blend of chitosan and MOS extract for settling and removing cyanobacterial biomass. Furthermore, we also examined whether or not the effectively treated reservoir water was capable of supporting the growth of cyanobacteria. The growth experiment was expected to give an insight into the longevity of the intervention method.

2. Materials and methods

2.1. Study area

Legedadi Reservoir is one of the major drinking water sources for the capital city of Ethiopia, Addis Ababa. It is administered by Addis Ababa Water and Sewerage Authority [21]. The reservoir is located at an altitude of 2450 m a.s.l. and at a geographical position of 90 01’ - 90 13’ N latitude and 380 60’ - 390 07’ E longitude (Fig 1). The reservoir, was constructed in 1967, with an initial storage capacity of 45.9 Mm3 and a surface area of 5.324 km2 [21]. It has a total catchment area of 205.7 km2. It has a mean and maximum water depth of 4 m and 30 m, respectively. The reservoir has an outflow rate to the water treatment plant of 126,666 m3/day and a water retention time of 325 days [21].

Fig 1

Map showing the location of the Legedadi Reservoir.

2.2. Reservoir water, chemicals and other materials

The water sample used for the experiment was collected around the dam in January, 2018. During sampling month, the mean concentration (μg/L) of total phosphorous (TP) and soluble reactive phosphate-P (SRP) were 597 and 167 respectively. While the mean concentration (μg/L) of nitrate-N and ammonium-N were 276 and 3.06 respectively. In the reservoir, the mean concentration (mg/L) of total suspended solids (TSS) was 390 and the mean concentration (mg/L) of total dissolved solids (TDS) was 77. The mean value of pH and conductivity (K25, μS cm−1) were 7.9 and 29.9 respectively. Cyanobacteria constituted 98% of total phytoplankton taxa, with species of Dolichospermum and Microcystis dominating the phytoplankton assemblages [22].

Dry Moringa oleifera seeds (MOS) with the pod, a natural coagulant, were purchased from Addis Ababa (Ethiopia). According to [23], MOS obtained from Ethiopia, on a dry matter basis of (g/100g) had the chemical composition of 6.1 ± 0.2, moisture content, 41.4 ± 1.6, oil content, 42.6 ± 1.4, protein content, 5.1 ± 0.3 crude fiber content, and 55.6 ± 1.5 crude protein content.

The healthy seeds (about 1.0 cm long) were selected after they were de-shelled and oven-dried at 105°C for 2 hrs. The kernels were crushed in a mortar and sieved through 300 μm to produce a powder with particles of ∼300 μm diameter [24]. The powder was stored in an airtight container and used within a month. To extract the active cationic coagulating proteins, 5 g of the seed powder was suspended in 100 mL of 1.0 mol/L NaCl solution and stirred using a magnetic stirrer for 30 min. The solution (MOS modifier) was filtered through a membrane microfiber filter paper of 0.45 μm pore size, yielding a stock of 2.8 ± 0.032 g/L of MOS extract. An additional stock of 100 mL of 1.0 mol/L NaCl Milli-Q water was prepared as a control [24,25].

The coagulant Chitosan was obtained from Polymar Ciência e Nutrição S/A (Ceara`, Brazil). First, in 200 mL flasks, 100 mg chitosan was dissolved in 20 mL Milli-Q water. Then, the solution was acidified by adding 100 μL of 96% acetic acid (Merck analytical grade). The solution was diluted to 100 mL with Milli-Q water, which yielded a stock of 1 g/L chitosan modifier. An additional stock of 100 μL of 96% acetic acid was added to 100 mL Milli-Q water and used as control [26]. The coagulant poly aluminium chloride (PAC, Aln(OH)mCl3n-m, density 1.37 kg/L, 8.9% Al, 21.0% Cl) was provided by Dr G. Waajen (Regional Water Authority Brabantse Delta).

2.3. Floc and sink assays

The ‘floc and sink’ assay was performed using different doses of coagulants/flocculants to coagulate and settle cyanobacterial species found in samples from Legedadi Reservoir. The reservoir has a mean annual turbidity (NTU) of 433 and exhibited a year-round occurrence and dominance of Microcystis aeruginosa and Anabaena (currently known as Dolichospermum) spp.

Initially, to maintain the growth of cyanobacteria, WC medium with vitamins added (H, biotin, and B12, cyanocobalamin, at 50 ng/L and B1, thiamine HCL, at 100 ng/L) [27], was added to the reservoir samples.

At the start of the experiment, chlorophyll-a (Chl-a) of 130 μg/L was recorded and cyanobacteria were in good condition as reflected by their Photosystem II efficiency (PSII) of 0.66 (± 0.01).The initial Chl-a concentration (μg/L) and PSII efficiency were determined using a phytoplankton analyzer (PHYTO-PAM, Heinz Walz GmbH, Effeltrich, Germany).

All experiments were conducted as follows: 11 mL Legedadi Reservoir sample was transferred into 15 mL glass tubes. Samples were treated with the coagulants/flocculants (PAC, MOs, chitosan, an effective dose combination of chitosan and MOS) or left untreated (controls). After dosing, the contents in each test tube were mixed briefly using a glass rod. Then, the samples were placed on a laboratory bench at room temperature and under still conditions [26].

After 1 h, 2 mL samples were taken from the top and bottom of the tubes using a 10 mL Eppendorf reference pipet. The 2 mL sample was used for the determination of Chl-a concentration and PSII efficiency as a measure of the health of the cyanobacterial cells. Then, the 2 mL sample was diluted 10 times with Milli-Q water and turbidity was measured using a turbidity meter (Hach 2100 P). The pH was measured in the glass tubes using a WTW pH meter.

PAC was dosed at 0, 1, 2, 4, and 8 mg Al/L and Chitosan was dosed at 0, 1, 2, 4, and 8 mg/L, while MOS extract was first dosed at 0, 10, 50, 100, 140 mg/L, and then the dosing was narrowed to a range of 0–50 (0, 10, 20, 30, 40, & 50) mg/L. Immediately after dosing, the contents in each test tube were mixed briefly using a glass rod. In all the four series of experiments, controls, and treatments were run in triplicate. Tubes were left untouched for 1 h, where after 2 mL top and 2 mL bottom samples were taken and analyzed as outlined above.

2.4 Treated reservoir water and its effect on cyanobacterial re-growth

In the second experiment, the effect of treated reservoir water (after treatment with an effective dose of MOS (30 mg/L) on cyanobacteria growth was assessed. This experiment aimed to test the hypothesis that the treatment lowered turbidity of the reservoir water (RW) to such an extent that light would not be limiting to cyanobacteria. To this end, a 26 days growth experiment was conducted using three cyanobacterial cultures: Microcystis aeruginosa PCC 7820, obtained from Pasteur Culture Collection (Institute Pasteur, Paris, France); Anabaena flos-aquae SAG 30.87 obtained from the Culture Collection of Algae at Gottingen University (Germany) and a Microcystis sp. culture (Microcystis sp. which was collected and isolated from Legedadi Reservoir, Ethiopia).

The three cyanobacteria were cultured in triplicate using three media, namely, modified WC medium [26], unaltered (raw) reservoir water (RRW) and treated reservoir water (TRW). In total, 27 replicates were used in this experiment. The experiment was conducted in 100 mL sterile Erlenmeyer flasks to which either 50 mL of TRW, 50 mL RRW, or 50 mL of autoclaved and cooled WC medium was added. Flasks with TRW or RRW received nutrients similar to those in the WC medium.

Then an inoculum of cyanobacterial cultures with Chl-a concentration of 20 (± 0.1) μg/L was added to the designated flask. One flasks with 50 mL RRW without addition of inoculum was included as additional control. Flasks were closed with a cellulose plug and placed at random in a Gallenkamp ORBI-SAFE Netwise Orbital Incubator at 25°C, 40 rpm shaking and in a 12:12 h light: dark cycle. The light: dark cycle was programmed in such a way that light intensity increased gradually to a maximum of 130 μmol quanta m-2 s-1 and subsequently decreased again to darkness, which resulted in daily average light intensity of ∼65 μmol quanta m-2 s-1. The light intensity within the incubator was measured at 22 locations and the experimental flask was swirled every time before sampling.

Samples were taken from the cultures initially and after 1, 2, 3, 6, 8, 11, 14, 19, and 26 days and were analyzed for Chl-a concentration and PSII efficiency using the PhytoPAM.

2.5 Data analysis

To examine the differences in the efficacy of coagulants/flocculants, one-way ANOVA at 95% confidence level was used. Significant differences were determined using a Tukey post hoc comparison test (p < 0.05). As data on PSII efficiency failed normality test, Kruskal-Wallis One Way Analysis of Variance on Ranks was run instead. As growth rate also failed the normality test, Friedman Repeated Measures Analysis of Variance on Ranks was made to determine whether there was a significant difference among cyanobacteria and growth media. Significant differences were determined using a Tukey post hoc comparison test (p < 0.05).

The Chl-a data over time were analyzed per replicate by fitting the logistic growth model in which the rate of ‘population increase’ (dA/dt) is a function of the population density determined by Chl-a (A) and two parameters, the growth rate r and the carrying capacity K:

The analytical solution: was added as user-defined equation to the Dynamic Fit Wizard in the program Sigma Plot, version 14.0, and growth rates were determined by iterative non-linear regression. Growth rates were compared by one-way ANOVA for TRW and WC medium, as no reliable estimates could be obtained for RRW, and therefore the preferred test, a two-way ANOVA with cultures inoculated and medium type (TRW, RRW, WC) as fixed factors, was not performed.

3. Results

3.1. Determination of optimal dose of flocculants

The turbidity of the reservoir combined with different coagulants (chitosan, PAC, MOS and a combination of MOS and chitosan) resulted in the settling of cyanobacteria after 1h treatment period (Fig 2).

Fig 2

Cyanobacterial removal efficiency of different flocculants in floc and sink experiment.

Chlorophyll-a concentrations (μg/ L) in the top 2 mL (top light gray bars) and bottom 2 mL (lower dark gray bars) of cyanobacteria suspensions incubated for 1 h with different concentrations of flocculants: A) chitosan B) MOS & C) PAC. For chitosan, MOS and PAC series, open triangle represents the pH value. Closed and open circle represents the top and bottom PSII efficiency values.

In the chitosan series, a concentration of 1 and 2 mg/L reduced Chl-a in the top of the tubes by 13% and 23%, respectively. Chitosan doses of 4 and 8 mg/L lowered the Chl-a concentration in the top of the tubes by 59.5% and 86.1%, respectively, while in the bottom of the tubes, the Chl-a concentration increased 6.2 and 8.2 times respectively (Fig 2A).

At all doses of chitosan, pH and PSII dropped only gradually. In the top of the tubes, turbidity was reduced by 40.5% and 43.5% at doses of 1 mg/L and 2 mg/L chitosan, respectively. While, at a doses of 4 mg/L and 8 mg/L chitosan, the turbidity was reduced by 62.1% and 77.1%, respectively. In contrast, at the bottom of the tubes, at chitosan doses of 4 mg/L and 8 mg/L the turbidity was increased 2.4 and 3.5 times, respectively (Fig 3A). Hence, chitosan concentration of 4 mg/L was considered the effective dose without affecting the pH and PSII.

Fig 3

Effect of different flocculants on reservoir turbidity.

Level of turbidity (NTU) in the top 2 mL and bottom 2 mL of cyanobacterial suspensions incubated for 1 h with different concentrations of flocculants: A) chitosan top light gray chitosan bottom dark gray B. MOS top light gray and MOS bottom dark gray C) PAC top light gray and PAC bottom dark gray.

In the MOS series, doses of 20 and 30 mg/L reduced Chl-a sharply in the top of the tube by 79% and 93%, respectively. At the top of the tube, MOS concentrations of 40 mg/L and 50 mg/L reduced Chl-a further to 92.6% and 95%, respectively. In contrast, at the bottom of the tubes, the MOS doses of 30 mg/L, 40 mg/L, and 50 mg/L increased Chl- a levels 2.7, 2.9, and 3.5 times (Fig 2B). At 20 mg/L MOS, PSII was reduced from 0.52 to 0.35, at 30 mg/L then afterward, remained around 0.35, while PSII dropped further to 0.26 and 0.23 at doses of 40 mg/L and 50 mg/L respectively. The pH dropped only gradually from pH 9.12 to pH 8.05 (Fig 2B).

In the top of the tubes, MOS concentration of 20 mg/L and 30 mg/L reduced the turbidity by 67% and 79%, respectively, whereas in the bottom of the tubes, turbidity increased by 6 and 6.8 times, respectively (Fig 3B). Therefore, MOS concentration of 30 mg/L was considered the effective dose without affecting the pH and PSII.

In the PAC series, at concentrations of 1 and 2 mg Al/L, Chl-a in the top of the tubes remained unaffected. PAC concentrations of 4 and 8 mg Al/ L, reduced Chl-a concentrations in the top of the tubes by 24% and 30%, respectively, while in the bottom of the tubes, the Chl-a levels increased 1.2 and 1.4 times (Fig 2C). At PAC dose of 1 mg Al/L, PSII efficiency dropped from the control (0.79) to 0.64, whereas at PAC dose of 2, 4 and 8 mg Al/L, PSII efficiency remained unaffected, indicating that the performance of cyanobacteria was not affected by increasing PAC doses with pH remaining unaffected at all doses.

In the top of the tubes, PAC doses of 4 mg Al/L and 8 mg Al/L reduced the turbidity by 52% and 56%, respectively, while in the bottom of the tubes, the turbidity increased by 1.7 and 2.0 times, respectively (Fig 3C). Hence, PAC concentration of 4 mg Al/L was considered an effective dose without affecting the pH and PSII efficiency.

3.2. The efficacy of coagulants in removing cyanobacteria from the reservoir water

At 30 mg/L MOS, most of the cyanobacterial cells were forced to sink to the bottom of the tubes, while the top layer of the tubes became clear (Fig 4). MOS was the most effective coagulant. One-way ANOVA indicated that Chl-a concentrations among treatments were significantly different for both the top (F4,14 = 179.8; p < 0.001) and the bottom (F4,14 = 323.0; p < 0.001) of the test tubes. Likewise, the combination of 4 mg/L chitosan and 30 mg/L MOS was highly efficient in clearing the top water layer and settling most of the cyanobacteria to the bottom of the tubes. Compared to other coagulants, PAC showed the least cyanobacteria removal efficiency (Fig 4).

Fig 4

The clearing effect of flocculants on the reservoir water.

Chlorophyll-a concentrations (μg/ L) in the top 2 mL (top light gray bars) and bottom 2 mL (lower dark gray bars) of 130 μg/L cyanobacterial suspensions incubated for 1 h with different concentrations of the flocculants: PAC (4 mg Al/ L), chitosan (4 mg/L) and MOS (30 mg/L). Also included are the pH values (open triangles), PSII efficiency in the top (closed circles) and bottom (open circles) of the experimental tubes.

PSII efficiency remained fairly stable in all treatments (chitosan, MOS and PAC) in both the top and bottom of the test tubes. Kruskal-Wallis One Way Analysis of Variance on Ranks indicated that differences in PSII among treatments were significant for the top (H4 = 11.249; p = 0.024), but not for the bottom (H4 = 8.841; p = 0.065) of the test tubes. The pH showed only minor fluctuations between pH 6.7 and 7.3 (Fig 4).

3.3 The effect of treated reservoir water on cyanobacterial re-growth

The median Chl-a concentrations of different cyanobacterial species (Anabaena flos-aquae, Microcystis aeruginosa, and Microcystis sp. culture from Legedadi Reservoir) grown in different media are shown in Table 1.

Table 1

Variations in chlorophyll-a (μg L-1) levels among experimental treatments.

Treatment	Median	Interquartile range (IQR)
TRW culture(Microcystis sp.)	61.4	19.5–1888.9
TRW Anabaena flos-aquae	57.8	20–1336.2
TRW Microcystis aeruginosa	35.6	18–1218.3
Modified WC culture(Microcystis sp.)	306.5	37.6–554.4
Modified WC Anabaena flos-aquae	46.6	11.9–696.7
Modified WC Microcystis aeruginosa	111.5	25.6–1446.7
RRW culture(Microcystis sp.)	389.3	175.9–494.4
RRW Anabaena flos-aquae	382.3	170–557.1
RRW Microcystis aeruginosa	342.6	162.3–726.5

Overall, cyanobacterial species did not show any sign of growth during the first 3 days of the experimental period. In TRW, all cultures showed a period of about 10 days with slow growth, where after growth accelerated (Fig 5A). One-way ANOVA indicated no differences in growth rates among cultures (F2,8 = 1.653; p = 0.268). The overall average growth rate was 0.39 (± 0.16)/day. In WC medium, the cyanobacteria started growing earlier than in TRW, but did not reach the high biomass as in TRW after 26 days (Fig 5B). The cultures with Microcystis PCC7820 added (labelled ‘Microcystis’) reached carrying capacity after 14 days, Anabaena started to decline after 14 days, while Microcystis from Legedadi (labelled ‘Culture’) started with slower growth, but reached higher Chl-a concentration at the end of the experiment (Fig 5B). One-way ANOVA indicated significant differences in growth rates among cultures (F2,7 = 29.839; p = 0.002). All Pairwise Multiple Comparison Procedures (Holm-Sidak method) revealed that growth rates of all cultures were significantly different from each other (p < 0.05). Growth rate of the culture was highest with 1.30 (± 0.24)/day, that of Microcystis PCC7820 was 0.64 (± 0.16)/day and of the Anabaena cultures it was 0.25 (± 0.6)/day. Iterative regression on data of one of the Legedadi Microcystis incubations did not converge (r2 = 0.186), hence, no growth rate of that replicate was determined. In RRW, all incubations showed a decline in Chl-a after 14 days. The logistic model only yielded poor fits (r2 = 0.131–0.640) and no reliable growth rates could be determined (Fig 5C). The RRW without any cyanobacteria added showed a growth rate of 0.22/day (r2 = 0.940).

Fig 5

Growth of cyanobacterial species in different medium.

Microcystis sp.culture from Legedadi Reservoir (Culture), Anabaena flos-aquae, and Microcystis aeruginosa in different media incubated for one month in A, Treated Reservoir Water (TRW) B, modified WC growth medium, and C, Raw Reservoir Water (RRW).

4. Discussion

4.1. The effect of coagulants/flocculants on the cyanobacteria

The results of this study are in line with the hypothesis that adding coagulants to water from the turbid Legedadi Reservoir could effectively remove cyanobacteria and suspended solids from the water without the need for adding additional ballast material. However, this water clearing effect was only observed when chitosan, MOS or their combination was used, but rather unexpectedly, not with PAC.

In this study, PAC doses of 4 and 8 mg Al/L were ineffective in coagulating and settling the cyanobacterial species in the reservoir’s water samples. In contrast, some previous studies showed that even lower doses of PAC (1 and 4 mg Al/L) effectively formed flocs of buoyant cyanobacteria [20,28]. The most likely reason for the low coagulation efficiency of PAC in the current study could be the relatively high pH (9.12–9.24) of the water. The observed higher pH was a physical state commonly associated with cyanobacterial blooms [29]. A high pH, along with high alkalinity may hinder the coagulating efficiency of PAC, thereby affecting its ability to effectively remove cyanobacterial species from the water bodies [30]. A similar observation was made by [19], who observed that large flocs were only formed at PAC doses of > 8 mg Al/L in water that had a high pH (pH = 10), as those higher PAC doses were able to reduce the pH of the water to levels that allowed formation of aluminium hydroxide flocs. Hence, it is likely that higher PAC doses of > 8 mg Al/L could also have been effective in Legedadi Reservoir water. However, due to potential health concerns of aluminium in finished drinking water, the World Health Organization (WHO) recommends to minimize aluminium levels in water to 0.1–0.2 mg/L [31]. Thus, additional experiments can be recommended to test PAC coagulation at a higher dose, but those should also include estimates of residual, dissolved organically bound Al [32].

Organic coagulants, such as chitosan, are viewed as safer, non-toxic and eco-friendlier coagulants than metal-based coagulants like PAC [33]. Different studies have revealed that chitosan is an effective bio-coagulant used in the removal of cyanobacteria from water [19,20,24]. In Legedadi Reservoir water, chitosan removed cyanobacterial species effectively at a dose of 8 mg/L despite the prevalence of moderately high pH. Some studies have reported that pH influences chitosan coagulation properties [34,35], and that high pH hampers the coagulation [36]. In this study, pH was not above 9 in the chitosan treatments, and at a dose of 8 mg/L pH was even reduced to almost 8. Clearly, pH was somewhat lower than that in the PAC treatment (see Fig 2), which urges for care in comparing the efficacy of the different coagulants. Chitosan may also cause cell lysis in cyanobacteria [37,38], therewith releasing cyanotoxins to the water [19]. In our study, no indications for rapid cell lysis were observed, no increase in chlorophyll-a signal was detected, and no strong decline in PSII efficiency to value of or close to zero were measured [38].

The other natural coagulant used in this study, Moringa oleifera seed extract (MOS) also removed turbidity and cyanobacteria from the reservoir water. This is in agreement with other studies that showed MOS was effective in flocculating and removing cyanobacteria from water [24,39]. MOS, dosed at 50 mg/L, could reduce up to 90% of the turbidity and the Microcystis aeruginosa Chl-a concentration [40]. MOS along with chitosan removed M. aeruginosa cells effectively in freshwater, and Amphidinium carterae and Chlorella sp. in seawater [24]. Other studies also reported MOS efficiency in removing harmful species of Microcystis [25,41]. In the current experiment, 30 mg/L MOS was effective in removing cyanobacterial species without observable cell lysis as PSII efficiency was only marginally affected.

This effective dose of MOS is relatively lower than the doses used in several other studies e.g. [40,42] due to the high turbidity of the reservoir. According to [40,42] the optimum dose of MOS is reduced as the water turbidity increases. The ability of MOS to flocculate the algal cells is ascribed to the interaction between the cells and the active protein molecule in the MOS. The cyanobacteria behave like negatively charged particles because of chemical materials comprising the cell walls, while the active cationic protein molecule in the MOS acts as positively charged particle that interacts with the cell wall and flocculated the cyanobacteria [43].

At higher concentrations of 50 mg/L, MOS and above, there was a sign of potential cell lysis with a stronger reduction of PSII efficiency. When a considerable proportion of the cells are lysed, a sharp drop in PSII-efficiency could be expected [44]. Therefore, using MOS at lower dose is crucial since MOS is known to be (more) effective in removing cyanobacteria, but less effective in removing dissolved toxins such as microcystins [42], although a recent study found up to almost 55% removal of microcystins by MOS [41].

The observed decline in PSII efficiencies indicated that there is some detrimental effect of MOS on the cyanobacteria occurring in the relatively short duration of the experiments. However, previous growth experiments with MOS have revealed that growth of M. aeruginosa is not hampered at 32 mg/L, while it was strongly reduced at 64 mg/L [45]. Together with findings of [46], who reported that at doses of 20–160 mg/L MOS, M. aeruginosa populations died, which was corroborated by a rapid drop in PSII efficiency to zero. Higher dose of MOS is not recommended. At high dose, the flocculation effect of MOS extract is reduced owing to charge neutralization, which results in charge reversal and destabilization of the cyanobacterial cells [39]. Some potential damage, causing a delayed effect on M. aeruginosa in settled flocs, has been observed for chitosan [47]. Such delayed effect can be viewed as beneficial, because damaging and gradual lysis of settled cyanobacteria (within a few days) will minimize the risk of resuspension and recolonization of the water column. Moreover, cyanotoxins will be degraded rapidly by the decomposing bacteria living near the sediment [48].

The relatively high pH observed in this experiment did not show major influence on the flocculation capacity of MOS, which is supported by findings in other studies that also concluded cyanobacterial removal efficiency of MOS is not affected by pH in the range of pH 5–9 [25,49]. Hence, MOS could be a good alternative for coagulating and settling cyanobacteria in water bodies that exhibit high pH. However, as the isoelectric point of the coagulation proteins of MOS is around pH 10 [50], pH 10 seems to be the upper limit of MOS use.

4.2. Floc and sink techniques of cyanobacteria removal

The results of this study show that in tropical turbid and moderately high pH reservoirs/lakes, low- dose biodegradable and environmentally safe coagulants such as, MOS, chitosan or a combination MOS, and chitosan is a good alternative for effectively settling harmful cyanobacteria out of the water column. The results also indicated that the turbidity of the reservoir, which is a result mostly of suspended solids, could be a good substitute for external ballast material in settling cyanobacteria.

Previous studies used a ‘floc and sink’ technique in which ballast material was introduced before the addition of an effective dose of coagulant [13,14,19,20,24,26,51].

The rational of testing coagulants using the reservoir turbidity as ballast in floc and sink techniques has several advantages: 1) restoration cost would be cheaper as it will reduce the cost of adding external ballast material, 2) the turbidity will be reduced, and the reservoir water will regain its transparency allowing increased resilience against disturbances, and 3) drinking water treatment cost will be reduced.

The fact that this experiment was conducted using reservoir water samples makes the results of this investigation more realistic. The present results are important in combating positively buoyant, surface scum-forming cyanobacteria that present one of the greatest threats to the well-being of humans and animals [52]. Besides, intervention strategies like using algaecides that cause liberation of toxins from cells are not the preferred management options as they eventually lead to the exposure of the public to cyanotoxin [9]. Thus, water supply managers should reconsider the use of algaecides in drinking water source water bodies and replace them with effective and environmentally safe natural flocculants like MOS. MOS can also reduce the concentration of extracellular cyanotoxins (microcystins) by 50% [41]. However, there are concerns regarding the use of biodegradable flocculants like MOS. MOS availability is limited to certain regions with tropical and subtropical climates [53]. Furthermore, Moringa oleifera is a multipurpose tree. Thus, its use in the removal of cyanobacteria leads to a conflict of interest. Besides, applying MOS in the reservoir might increase the organic load of the reservoir, thereby affecting the level of DO [54]. Moreover, high doses of MOS not only increase organic carbon, but also nutrients that can promote microorganisms growth and cause odour, taste, and colour problems [55,56].

Therefore, applying a minimum dose of MOS is preferable. MOS combined with chitosan will reduce the amount of MOS needed for water restoration. Therefore, MOS combined with chitosan can be a good alternative coagulant for reservoirs/lakes in the tropical region and more up-scaled experiments can be considered.

4.3 Effect of Moringa oleifera in removing and preventing re-growth of cyanobacteria

The results of our growth experiments indicated that the low dose of MOS is not only efficient in removing buoyant cyanobacteria from the reservoir water, it also delayed the re-growth of common bloom-forming cyanobacterial species such as Microcystis aeruginosa and Anabaena flos-aquae. During the first 3 days of the experimental period, the growth of the species in all media was negligible. This delay in cyanobacterial growth could be due to the need for acclimatization through biochemical adjustment [57]. The prolonged acclimatization in TRW (indicated with “TRW” in Fig 5) was unexpected and in contrast to our hypothesis as in the new clear water state, nutrients were believed to enhance the growth of cyanobacteria (20 μg/ L) inoculum. The delay could be a result in of inhibitory factors or anti-cyanobacterial action of MOS [46]. However, within the course of the experiment, cyanobacterial Chl-a concentrations reached much higher values in TRW than in either the raw reservoir water (RRW) or in the artificial growth medium (WC medium). A higher biomass of carrying capacity in the TRW treatment may be caused by additional nutrients added from MOS, which contains phosphate, nitrate, ammonium [46]. The much lower growth and even decline after 14 days observed in RRW could have been caused by higher turbidity, yet the single RW control seems to contradict this. The experiment cannot provide an answer why Chl-a in the RW was much higher than in RW to which cyanobacteria were added (RRW treatment). Clearly, more research will be needed, nonetheless, the experiment provides indications that clearing the reservoir with ongoing nutrient inputs may come with a risk that cyanobacterial biomass will reach much higher concentrations after in-situ coagulation and sinking than without. Up-scaled experiments are needed to get insight in the longevity of the intervention and possible drawbacks as outlined above.

These experimental results indicate the potential applicability of Moringa oleifera seed or a combination of Moringa oleifera seed and chitosan as flocculants without adding ballast material in removing cyanobacterial species in turbid lakes/reservoirs.

Moringa oleifera seed seems to cause a short-term delay in re-growth of cyanobacteria after treatment of tropical lakes/reservoirs.

Floc and sink experiments were conducted effectively in a small sample volume (11 mL) of reservoir/lake water.

Up-scaled experiments are needed.

Experimental set-up of the Floc and Sink technique: (A) conceptual diagram, (B) Coagulation experiment with different coagulants (C) Top and bottom 2mL samples after 1hr experiment.

(TIF)

Click here for additional data file.

One way ANOVA analysis on effect of coagulants on cyanobacterial species.

(DOCX)

Click here for additional data file.

Descriptive statistic of growth of cyanobacterial species on different growth medium.

(DOCX)

Click here for additional data file.

Iterative non-linear regression analysis of cyanobacterial growth at different medium using a logistic growth model.

(XLSX)

Click here for additional data file.

15 Dec 2020

PONE-D-20-31065

Removal of cyanobacteria from a water supply reservoir by sedimentation using natural flocculants and water turbidity as ballast: Case of Legedadi Reservoir (Ethiopia)

PLOS ONE

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Demeke Kifle

Seyoum Leta

Maíra Mucci

Miquel Lürling '

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Demeke Kifle

Seyoum Leta

Maíra Mucci

Miquel Lürling '

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Reviewer #2: Yes

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Reviewer #1: This study investigated the removal of cyanobacteria from a water supply resevoir by sedimentation using natural flocculants and water turbidity as ballast. This is a meaningful study, however, this manuscript is still not in a publishable state. The manuscript needs to be further improved in research design and analysis. And some sentences in this manuscript are hard to understand, it needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure so that the goals and results of the study are clear. Furthermore, some important aspects of this study are confusing.

1. Title: “use water turbidity as ballast”, but there’s no experimental design on how to use the turbidity of water as the ballast in this paper.

2. Grammar check: such as page 5, line 88-90; page 7, line 139-141……

3. Page 5, line 90-91: “Legedadi Reservoir is a very turbid reservoir”, how high is the turbidity? “the high turbidity was considered as a unique feature and expected to act as ballast”, does the high turbidity reservoir only be used as a case study? It should be designed to compare the effects of different turbidity.

4. Section 2.1: The water quality of the reservoir is not explained. Water quality is an important condition for the research, such as the turbidity of the reservoir when the experiment was carried out, the dominant algae and algae concentration, etc.

5. Page 7, line 149: “After dosing, the contents in each test tube were mixed briefly using a glass rod.” It is confusing. Coagulation-flocculation experiments were usually performed by jar test with a six-paddle stirrer. How to control the specific conditions of the mixing operation using a glass rod, so whether the experimental results of the glass rod mixing would be affected by the man-made operation errors?

6. Page 8, line 161-163: The dose design of PAC, Chitosan, and MOS was different, would the difference of coagulation effect be affected by the dosage range of different coagulants? For example, the dose of 8mg/L chitosan as shown in Fig. 3 has not been proved to reach the optimal dose with stable removal efficiency, while the span of MOS dose is larger with 20 mg/L has a relatively high efficiency and 30mg/L has reached the optimal dose.

7. Section 2.4: It is not recommended to use question sentence as subtitle in the experimental methods section.

8. Page 11, line 233-234: What does the “drop” and “remian” of PSII indicate? It should be explained.

9. Page 12, line 253: It is recommended to list the process and results of data analysis in a table or supporting information, and the supporting information in this paper seems to have uploaded the wrong file.

10. Page 13, line 288: The “Linear regression model” is recommended to provide in detail.

11. Page 14, line 297: “8Al/L”, check unit.

12. Page 15, line 330-331: “At higher concentrations of 50 mg/L MOS and above, there was a sign of cell lyses with a high reduction of PSII efficiency”. However, Fig. 3 showed that PSII efficiency had dropped when the MOS dose is 20 mg/L. Confusing.

13. Page 16, line 345: Section 4.2, check subtitle.

14. Fig. 2 is not clear, and there is no directional significance of the actual results. What is the author’s intention to put Fig. 2 ?

15. Fig. 3, “Moringa” in Fig.3B should be consistent with “MOS” in the text.

16. Fig.4, why not put MOS results in the same figure with PAC and chitosan? Using the drawing method of Fig. 3 to distinguish the top and bottom is a better choice.

17. Fig.6 is not clear.

18. References: It would be better to quote more recent references such as in the last three years.

Reviewer #2: The manuscript evaluated the efficiency of the flocculants/coagulants chitosan, Moringa oleifera seed (MOS) and poly-aluminium chloride (PAC) in settling cyanobacterial species in Legedadi Reservoir. The authors suggest that the efficacy of a flocculant/coagulant in the removal of cyanobacteria is influenced by the uniqueness individual lakes/reservoirs, implying that mitigation methods need to consider the unique characteristic of the lake/reservoir.

The manuscript was generally well organized, and the conclusion is constructive to remove cyanobacteria and control the blooms.

Comments for revision and correction:

1) Introduction: the authors give more information and description to support their assumption: water turbidity functions as ballast.

2) The chemical composition of Moringa oleifera seed may change between those collected from different locations. This information is helpful to understand its efficiency.

3) In Fig.3 A &C，Chla concentration was higher in the high treatments than in the control for bottom and surface?

4) In Fig.5，treated with MOS，PSII efficiency decreased at the surface, but increased for cyanobacteria at the bottom. Why?

5) As turbidity is assumed to be ballast, it is better to give more physical and chemical information of the turbidity. Know which kinds of turbidity function better.

6) In Fig.3, Fig.4, Fig.7, adding error bars.

7) Redrawing Fig.6 to be clear for readers.

8) The English needs correction. For example, Line 86: future =feature? Line 88-89: “The availability of local soils with natural P binding capacities was. For

instance, considered…” =” For instance, the availability of local soils with natural P binding capacities was considered… ”.

**********

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Reviewer #1: No

Reviewer #2: No

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8 Feb 2021

Responses to editorial requirements

COMMENT

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming

RESPONSE

All contents of the manuscript is revised according to PLOS ONE's style

COMMENT

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

'Hanna habtemariam

Demeke Kifle

Seyoum Leta

Maíra Mucci

Miquel Lürling '

b. Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

RESPONSE

Thank you for your comment. We removed funding related text information from the manuscript. Additional we specify our funding statements on the cover page

COMMENT

Please complete your Competing Interests statement to state any Competing Interests a. Please complete your Competing Interests statement to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now

b. This information should be included in your cover letter; we will change the online submission form on your behalf.

RESPONSE

Thank you for your comment and guidance we have included our Competing Interests statement on the cover letter

COMMENT

4. We note that Figure 1 in your submission contains map images which may be copyrighted. We require you to either (a) present written permission from the copyright holder to publish these figure specifically under the CC BY 4.0 license, or (b) remove the figure from your submission:

If you are unable to obtain permission please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

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Thank you for your comment. we removed figure 1 from the manuscript and replaced with new Figure

COMMENT

5. Please upload a new copy of Figure 2a as the detail is not clear. Please follow the link for more information: https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/

RESPONSE

Thank you for the comment we have uploaded a copy of figure 2a (in tiff format)

COMMENT

6. Please include a copy of Table 1 which you refer to in your text on page 13.

RESPONSE

Thank you for your comment. We have included Table 1 on page 13 line 299

Responses to Reviewer’s comments

Reviewer#1

COMMENT

Further improved in research design and analysis. And some sentences in this manuscript are hard to understand, it needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure so that the goals and results of the study are clear some important aspects of this study are confusing.

RESPONSE

Thank you for your critical review. We revised the entire manuscript with correction of grammar, spelling and by improving readability. The changes made in the manuscript were red-highlighted

COMMENT

Title: “use water turbidity as ballast”, but there’s no experimental design on how to use the turbidity of water as the ballast in this paper.

RESPONSE

Thank you for your comment. In the ‘floc and sink’ approach a ballast compound is first introduced where after flocculants/coagulants will be added to settle phytoplankton out of the water. In the current experiment, since the reservoir is turbid, because of high concentrations of suspended solids, we hypothesized that there would be no need of adding external ballast. Therefore, we designed an experiment applying only coagulants/flocculants, which were added to the reservoir water without any additional ballast. We agree that the term “water turbidity “ is not clear and hence we have rephrased the title emphasizing “suspended solids. We also have removed “natural” from the title as PAC cannot be viewed as a natural coagulant. The new title reads: “Removal of cyanobacteria from a water supply reservoir by sedimentation using flocculants and suspended solids as ballast: Case of Legedadi Reservoir (Ethiopia)”

COMMENT

Grammar check: such as page 5, line 88-90; page 7, line 139-140

RESPONSE

We accepted the comment and corrected the grammar on page 5, lines 85-91; page 7, lines 155-157

COMMENT

Page 5, line 90-91: “Legedadi Reservoir is a very turbid reservoir”, how high is the turbidity? “the high turbidity was considered as a unique feature and expected to act as ballast”, does the high turbidity reservoir only be used as a case study? It should be designed to compare the effects of different turbidity.

RESPONSE

We appreciated the comments provided by the reviewer. The annual mean turbidity of the reservoir was mentioned on page 7 line 142 of the original version of the manuscript. The turbidity in the reservoir is high with an annual mean turbidity of 433 NTU, and during the rainy season increasing up to 800–900 NTU. This is mostly due to suspended solids flowing in from the rivers and considered high enough to form easy coagulation with coagulants like MOS. Inasmuch as we’ve performed the experiments at Wageningen University, we were limited in the amount of Reservoir Water to be used. Indeed, with appropriate equipment it would be very nice to have a series of those experiments performed over the course of the year, since several water quality variables will change that could have an effect on removal efficiency.

COMMENT

Section 2.1: The water quality of the reservoir is not explained. Water quality is an important condition for the research, such as the turbidity of the reservoir when the experiment was carried out, the dominant algae and algae concentration, etc.

RESPONSE

We highly appreciate the comments. We have now included the concentrations of nutrients, pH, conductivity, and dominant algal groups encountered during the sampling time in section 2.1 (page 6, line 120–127). Information regarding algal concentration was included in the original version of the manuscript on page 8, line 158, while level of turbidity was indicated on page 7 line 153.

COMMENT

Page 7, line 149: “After dosing, the contents in each test tube were mixed briefly using a glass rod.” It is confusing. Coagulation-flocculation experiments were usually performed by jar test with a six-paddle stirrer. How to control the specific conditions of the mixing operation using a glass rod, so whether the experimental results of the glass rod mixing would be affected by the man-made operation errors?

RESPONSE

Thank you very much for your concern and comment. It is true that mixing is an important component of the coagulation process and in several coagulation studies, it was done in a jar with stirrer. However, in our experiment, we chose to opt for a more gentle mixing as has been done in most ‘floc and sink’ studies (e.g. Noyma et al., 2016, 2017; Miranda et al., 2017; de Lucena-Silva et al., 2019; Lürling et al., 2020). The rationale behind glass-rod stirring is that this mixing is more representative to the gentle mixing that often occurs in situ, while the standard jar test includes periods of vigorous mixing. Previous studies have shown that “mixing regime is not that crucial for flocs formation as long as there is slow mixing. Such mixing will also be the predominant regime in whole lake treatments” (Lürling et al., 2017).

de Lucena-Silva, D., Molozzi, J., dos Santos Severiano, J., Becker, V., de Lucena Barbosa, J.E., 2019. Removal efficiency of phosphorus, cyanobacteria and cyanotoxins by the “flock & sink” mitigation technique in semi-arid eutrophic waters. Wat. Res. 159: 262-273.

Lürling, M., Noyma, N., deMagalhães, L., Miranda, M., Mucci, M., van Oosterhout, F., Huszar, V.L., Marinho, M.M., 2017. Critical assessment of chitosan as coagulant to remove cyanobacteria. Harmful Algae 66: 1–12.

Lürling, M., Kang, L., Mucci, M., van Oosterhout, F., Noyma, N.P., Miranda, M., Huszar, V., Waajen, G., Manzi, M. 2020. Coagulation and precipitation of cyanobacterial blooms. Ecological Engineering 2020; 158: 106032.

Miranda, M., Noyma, N., Pacheco, F.S., de Magalhães, L., Pinto, E., Santos, S., Soares, M.F.A., Huszar, V.L., Lürling, M., Marinho, M.M., 2017. The efficiency of combined coagulant and ballast to remove harmful cyanobacterial blooms in a tropical shallow system. Harmful Algae 65: 27–39.

Noyma, N.P., de Magalhães, L., Furtado, L.L., Mucci, M., van Oosterhout, F., Huszar, V.L.M., Marinho,M.M., Lürling, M., 2016. Controlling cyanobacterial blooms through effective flocculation and sedimentation with combined use of flocculants and phosphorus adsorbing natural soil and modified clay. Wat. Res. 97: 26-38.

Noyma, N.P, de Magalhães, L., Miranda, M., Mucci, M., van Oosterhout, F., Huszar, V.L.M., Marinho, M.M., Lima, E.R.A., Lürling, M., 2017. Coagulant plus ballast technique provides a rapid mitigation of cyanobacterial nuisance. PLoS ONE 12(6): e0178976.

COMMENT

Page 8, line 161-163: The dose design of PAC, Chitosan, and MOS was different, would the difference of coagulation effect be affected by the dosage range of different coagulants? For example, the dose of 8 mg/L chitosan as shown in Fig. 3 has not been proved to reach the optimal dose with stable removal efficiency, while the span of MOS dose is larger with 20 mg/L has a relatively high efficiency and 30 mg/L has reached the optimal dose.

RESPONSE

We appreciate the constructive comment forwarded by the reviewer. We have chosen the dose of PAC and chitosan based on the results of previous studies. In those studies , strong reduction in PSII efficiency was found at a PAC dose of 4 mg/L or higher (Noyma et al., 2016), or 8 mg/L or higher (e.g. Miranda et al., 2017). Moreover, chitosan may also impair cell membrane integrity at higher doses (Mucci et al., 2017) and therefore, we did not include those higher doses in the tests performed here. Moreover, we hypothesized from the previous studies with freshwaters that a 2 mg Al/L and a chitosan dose of 2 mg/L would be effective. As regards MOS, we, however, learned that different studies used different, higher doses. Therefore, we did a two round dose estimation for MOS to estimate the effective dose . In the first round, we took the average values from different studies and evaluated doses of 0, 10, 50, 100, and 140 mg/L for their efficiency of cyanobacterial removal and cell lysis. Based on the results, we subsequently narrowed down the doses to 0, 10, 20, 30, 40, and 50 mg/L. We have added some sentences to the discussion to clarify our choice of test doses.

COMMENTS

7. Section 2.4: It is not recommended to use question sentence as subtitle in the experimental methods section.

RESPONSE

We accepted the comment and corrected the subtitle of section 2.4

COMMENT

8. Page 11, line 233-234: What does the “drop” and “remain” of PSII indicate? It should be explained.

RESPONSE

We have accepted the comment and added explanations for the “drop” and “remain” of PSII

COMMENT

9. Page 12, line 253: It is recommended to list the process and results of data analysis in a table or supporting information, and the supporting information in this paper seems to have uploaded the wrong file.

RESPONSE

We have accepted the comments and attached the results of data analysis as a supplementary file

COMMENT

10. Page 13, line 288: The “Linear regression model” is recommended to provide in detail.

RESPONSE

Thank you for this comment. It made us have a critical look at the data and that clearly revealed the growth observed was not following the exponential model. Hence, we have reanalyzed the data by iterative non-linear regression analysis using a logistic growth model. This provided far better estimates for growth rates in TRW and WC, but not in RRW where after 14 days a decline in Chl-a occurred. We have revised the methods, the results and the discussion accordingly.

COMMENT

11. Page 14, line 297: “8Al/L”, check unit.

RESPONSE

We accepted the comment and added the unit “mg ” to Al/L

COMMENT

12. Page 15, line 330-331: “At higher concentrations of 50 mg/L MOS and above, there was a sign of cell lyses with a high reduction of PSII efficiency”. However, Fig. 3 showed that PSII efficiency had dropped when the MOS dose is 20 mg/L. Confusing.

RESPONSE

We appreciated the critical comment. It is true that at MOS concentrations above 10 mg/L PSII efficiency started to decline from the initial value. However, compared to the dosage of 50 mg/L it was still considered marginal. We have rephrased the manuscript and also omitted the speculation on cell lysis as this was not measured. We also think that line 330–331 is confusing to readers. Therefore, we took out that sentence from the manuscript.

COMMENT

13. Page 16, line 345: Section 4.2, check subtitle.

RESPONSE

We accepted the comment and corrected the subtitle of section 4.2, page 354

COMMENT

14. Fig. 2 is not clear, and there is no directional significance of the actual results. What is the author’s intention to put Fig. 2 ?

RESPONSE

Thank you for your comments. Fig.2a was added with the intention of explaining the conceptual framework of the floc and sink assay. Fig. 2b shows the process of the floc and sink experiment and Fig.2c shows the level of treatment of effective coagulants. In figure 2c the right side picture shows the reservoir water before treatment, while the left side picture shows the reservoir water after treatment.

COMMENT

15. Fig. 3, “Moringa” in Fig.3B should be consistent with “MOS” in the text.

RESPONSE

We accepted the comment and adjusted Fig. 3B accordingly

16. Fig.4, why not put MOS results in the same figure with PAC and chitosan? Using the drawing method of Fig. 3 to distinguish the top and bottom is a better choice.

RESPONSE

We accept the comment and adjust Fig. 4 accordingly

COMMENT

17. Fig.6 is not clear.

RESPONSE

Thank you for the comment Fig. 6 shows the growth rate of different cyanobacterial species in different media during the 26 days experimental period. We have modified the graph using similar y-axis in each panel and we have included the non-linear regression outputs (logistic growth model).

COMMENT

18. References: It would be better to quote more recent references such as in the last three years.

RESPONSE

Thank you for your comment. Some old references were replaced with more recent ones

Reviewer #2

COMMENT

Introduction: the authors give more information and description to support their assumption: water turbidity functions as ballast.

RESPONSE

Thank you for your comment. We have accepted the comment and added information regarding turbidity as a ballast on page 5, lines 88-91

COMMENT

The chemical composition of Moringa oleifera seed may change between those collected from different locations. This information is helpful to understand its efficiency.

RESPONSE

We have accepted the comment and included information on the chemical composition of Moringa seed available in Ethiopia in the “Materials and methods” part .

COMMENT

3) In Fig.3 A &C Chla concentration was higher in the high treatments than in the control for bottom and surface?

RESPONSE

Thank you for your comment Chl-a concentration was higher only in the bottom. The addition of the coagulants caused settling of the phytoplankton and suspended solids leading to higher Chl-a in the bottom samples, but lower in the top compared to the controls.

COMMENT

4) In Fig.5，treated with MOS，PSII efficiency decreased at the surface, but increased for cyanobacteria at the bottom. Why?

RESPONSE

We appreciate your keen observation and question. The drop in PSII efficiency is to slightly above 0.4. In those two treatments, comparatively most cyanobacteria cells, approximately 96%, were found in the bottom with coagulants, which creates more bright conditions in the top. A general response of phytoplankton, including cyanobacteria, to rather rapid increased light intensity is a reduction of PSII efficiency (Deblois et al., 2013). In the bottom, there are no differences in PSII efficiency. Alternatively, the cyanobacteria remaining in the top were not precipitated with ballast weighed down flocs and thus potentially exposed to relatively more binding sites of Moringa (and chitosan) causing some damage.

Deblois CP, Marchand A, Juneau P (2013) Comparison of Photoacclimation in Twelve Freshwater Photoautotrophs (Chlorophyte, Bacillaryophyte, Cryptophyte and Cyanophyte) Isolated from a Natural Community. PLoS ONE 8(3): e57139. doi:10.1371/journal.pone.0057139

COMMENT

5) As turbidity is assumed to be ballast, it is better to give more physical and chemical information of the turbidity. Know which kinds of turbidity function better.

RESPONSE

We accepted the comment and provided some physicochemical features of turbidity

COMMENT

6) In Fig.3, Fig.4, Fig.7, adding error bars.

RESPONSE

Thank you for your comment, when we revised the documents we removed Fig 7. Moreover, estimation of the dose of coagulants shown in Fig. 3, and measurement of the turbidity shown in Fig 4, were based on only single trial results for each dose; Therefore, we were unable to add error bars to Fig. 3. and Fig. 4

COMMENT

7) Redrawing Fig.6 to be clear for readers.

RESPONSE

We have accepted the comment and have redrawn Fig. 6.

COMMENT

8) The English needs correction. For example, Line 86: future =feature? Line 88-89: “The availability of local soils with natural P binding capacities was. For

instance, considered…” =” For instance, the availability of local soils with natural P binding capacities was considered… ”.

RESPONSE

Thank you for your comments. The English of Line 86 and Lines 88-89 was corrected. We have also made proof reading and correction of the manuscript accordingly.

15 Mar 2021

PONE-D-20-31065R1

Removal of cyanobacteria from a water supply reservoir by sedimentation using flocculants and suspended solids as ballast: Case of Legedadi Reservoir (Ethiopia)

PLOS ONE

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Reviewer #1: There are still some problems to be corrected. It is not limited to the problems listed below. Please read the full text carefully and revise the details.

1) Page 4, line 86-87: Grammar check: “it’s” ?

2) Page 6, line 122: 276 µg/L what? Check your parentheses use.

3) Page 6, line 123-124: “The reservoir has a mean total suspended solids (TSS) concentration of 390 mg/L and total suspended solids” ? Check the statement expression and the use of parentheses.

4) Page 6, line 125-126: Sentence is unreasonable, lack of punctuation.

5) Page 6, line 128: Unpublished data is not recommended for citation.

6) Page 7, line 130-133: According to what? “moisture content of 6.1 ± 0.2, oil content of 41.4 ± 1.6,” and “protein content 42.6 ± 1.4,” ? Check your expression.

7) Fig.1 is not clear.

8) Fig. 2a, there are no relevant instructions in the text, and we still feel that Figure 2 has no actual data to point to value.

9) Fig.6 is not clear, the legend in the picture is invisible, and error bars are not clear.

Reviewer #2: I checked the revision of he manusscript, the authors have corrected and well responded to my questions.

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20 Mar 2021

Responses to Reviewer 1

COMMENT

There are still some problems to be corrected. It is not limited to the problems listed below. Please read the full text carefully and revise the details.

RESPONSE

Thank you for your comment. While we were addressing the comments we have made revision of the entire manuscript regarding grammar and punctuation, For easy identification of track changes in the manuscript, we red-highlighted in bold all changes made

COMMENT

Page 4, line 86-87: Grammar check: “it’s” ?

RESPONSE

Thank you for your comment. We have corrected the grammar

COMMENT

2) Page 6, line 122: 276 µg/L what? Check your parentheses use.

RESPONSE

Thank you for your comment. We have rephrased the sentence and corrected the use of parentheses

COMMENT

3) Page 6, line 123-124: “The reservoir has a mean total suspended solids (TSS) concentration of 390 mg/L and total suspended solids” ? Check the statement expression and the use of parentheses.

RESPONSE

Thank you for your comment we have rephrased the sentence and corrected the use of parentheses

COMMENT

4) Page 6, line 125-126: Sentence is unreasonable, lack of punctuation.

RESPONSE

Thank you for your comment we have added a punctuation mark in the sentence

COMMENT

5) Page 6, line 128: Unpublished data is not recommended for citation.

RESPONSE

Thank you for your comment. Currently, we have published the manuscript, therefore, this citation is published data.

COMMENT

6) Page 7, line 130-133: According to what? “moisture content of 6.1 ± 0.2, oil content of 41.4 ± 1.6,” and “protein content 42.6 ± 1.4,” ? Check your expression.

RESPONSE

Thank you for your comment we have rephrased the sentence

COMMENT

7) Fig.1 is not clear.

RESPONSE

Thank you for your comment. Fig 1 is the description of the study area. We believed that it is important to include a visual map for the reader for a better understanding of the study.

COMMENT

8) Fig. 2a, there are no relevant instructions in the text, and we still feel that Figure 2 has no actual data to point to value.

RESPONSE

Thank you for your comment. we have take-out figure 2 from the manuscript and put it to supplementary data

COMMENT

9) Fig.6 is not clear, the legend in the picture is invisible, and error bars are not clear.

RESPONSE

Thank you for your comment we have made all correction to Fig.6

24 Mar 2021

Removal of cyanobacteria from a water supply reservoir by sedimentation using flocculants and suspended solids as ballast: Case of Legedadi Reservoir (Ethiopia)

PONE-D-20-31065R2

Dear Dr. Habtemariam,

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Jean-François Humbert

Academic Editor

PLOS ONE

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Reviewers' comments:

26 Mar 2021

PONE-D-20-31065R2

Removal of cyanobacteria from a water supply reservoir by sedimentation using flocculants and suspended solids as ballast: Case of Legedadi Reservoir (Ethiopia)

Dear Dr. Habtemariam:

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