Effects of different cell states of Microcystis aeruginosa on coagulation process

Three different states of Microcystis aeruginosa, untreated cells, pure cells, and broken cells, were tested for coagulation efficiency to understand their effects on drinking water treatment facilities. Microcystis aeruginosa in its late log-growth phase to early stationary phase, day 12 of culture in the M11 medium, were collected since cells are the most active and reach its maximum population during those periods. Untreated cells samples showed worst results for turbidity removal while turbidities for pure cells and broken cells ones presented the similar levels with those for 0 cell samples after 3 Al-mg/L of coagulant dose. Organic matters from broken cells were not properly removed throughout coagulation processes since samples with organic matters untreated cells and broken cells showed higher levels of E254 and E260 after jar-tests than pure cell samples did. Overall, coagulation processes were highly interrupted when the Microcystis aeruginosa cells and organic matters from cells were co-existed. In addition, algae originated matters which may not remove by coagulation process could lead the secondary pollution when algae are introduced into drinking water treatment facilities during waterbloom. Therefore, the facility should pay attention on the results since they have to assure their water quality for public health.


Introduction
Urbanization and population growth lead increasing input of sewage and wastewater to the waterbodies and result in decreasing water quality, water pollution. Water pollution occurs when contaminants exceeding the capacity of the self-purification of water system and one of typical water pollution is eutrophication. The term "eutropic" is derived from the Greek word "rich in nutrients" and refers to the deterioration of water quality caused by excessive inorganic nutrients and/or organic matters in the waterbodies.
Phytoplankton and algae will dramatically increase once waterbodies are eutrophicated, Especially, cyanobacteria (blue-green algae) will be the dominant strain when temperature is high and this is so called the waterbloom. During waterblooms, cyanobacteria will cover the most of water surfaces and thihs causes blocking oxygen transfer between air and waterbody, perishing aquatic organism as well as generating unpleasant odor and taste. Some cyanobacteria, moreover, produce toxic substances such as hepatotoxin, nevertoxin, and cytotoxin which are causes of dermatitis, stomachache, headache and allergy of human being as well as the death of aquatic organism and mammals [1][2][3][4][5]. Furthermore, waterblooms by cyanobacteria are the cause of interrupting water treatment process, i.e. inhibiting coagulation process, shortening filter's longevity, increasing pH level, decreasing dissolved oxygen level, etc. [6][7][8].
Microcystis aeruginosa is the most common strain while waterblooms occur not only in Korea, but also in worldwide. In fact, toxic damages caused by Microcystis aeruginosa during waterblooms have been reported worldwide [9][10][11][12]. Microcystis aeruginosa produces microcystin, one of hepatotoxin, and the toxin may cause severe injury to the liver since it is already reported that hepatotoxins are the cause of the clinical consequences for acute or chronic liver injury, with the possibility of enhanced susceptibility to, and growth of, liver tumors [13]. Furthermore, Microcystis aeruginosa inhibit the water treatment process, especially for the coagulation process and filtration process [14][15][16]. Inappropriate managements, therefore, for Microcystis aeruginosa during drinking water treatment process may bring serious public health problems.
In this study, coagulation efficiencies for different algae cell status was investigated to understand whether coagulation inhibitions would be caused by algae cell itself or by organic matters originated from algae. The results would provide better ideas to water treatment facilities when waterbloom occurs in their water resources.

Cultivation of Microcystis Aeruginosa
Microcystis aeruginosa was obtained from Korean National Institute of Environmental Research and cultured in M 11 medium, most widely used for culturing Microcystis aeruginosa [17][18][19][20]. Table 1 shows the composition of M 11 medium. Microcystis aeruginosa were collected from its late log-growth phase to early stationary phase, specifically, day 12 of culture when algae reached their maximum cell numbers, approximately 3.4 x 10 8 cell/mL, since algae in this period should be the most active and release the most abundant organic matters. Followings are the cell counting process for the study; 0.1 mL of 12% NaClO was added in 100 mL of cultured water as well as coagulated water and left them one hour at room temperature as the pre-treatment. Water samples were sonicated with 30 W for 5 min to make cells fall apart after pre-treatment and then Hemocytometer, broadly used for microcell counting, were used to count cell numbers of Microcystis aeruginosa [21,22].

Preparing Different Cell States
Three different states of algae cells were prepared for the study; 1. untreated cells 2. pure cells 3. broken cells. Cells in M 11 medium were used as they were for untreated cell samples. Untreated cells contain pure cells and External Organic Matters (EOMs). Pure cells were obtained by centrifuging (4,000 g, 15 min) fully grown cells in their late log-growth phase to early stationary phase at M 11 medium. The centrifugation was repeated one more time after the supernatant from the first centrifugation was discarded and then samples were filled with distilled water. Broken cells were getting from destroying cell membrane by using high frequency sounds (20 kHz, 100 W, 10 min) and then samples were checked whether or not there might be unbroken cells with microscopic observation. The process was kept repeating until all cells were totally broken. Broken cell samples contain Internal Organic Matters(IOMs), Surface Organic Matters (SOMs), and EOMs [2].

Jar-Test
500 mL of water samples were used for jar-test. Distilled water was used to rule out the effects of other factors except algae cell itself. Turbidity and alkalinity of water samples were artificially adjusted using Kaolin and CaCO 3 ; Turbidity 40 NTU and Alkalinity 100 mg/L of CaCO 3 . Each state of Microcystis aeruginosa were added in water samples after turbidity and alkalinity were adjusted. Jar-tests were carried out with 140 rpm for first 10 min and 40 rpm for following 15 min and then water samples were left for 30 min to settle down flocks. Jar-tests were repeated with every 1 Al-mg increment of coagulant dose, 1 Al-mg to 18 Al-mg, for each state of cell sample and control one. Al 2 (SO 4 ) 2 ·16~18H 2 O (Alum) were used as coagulant and pH levels of tested water were adjusted to stay their levels at 7 ± 0.05 using 1 N and 0.01 N of NaOH and HCl during jar-tests.

Evaluation for Efficiency of Coagulation Process
Turbidity, UV 254 , UV 260 and residual aluminum were analyzed after each jar-test to evaluate efficiencies of coagulation process for removal of each cell state of Microcystis aeruginosa based on Drinking Water Quality Standard Methods [23]. Shimadzu UV spectrophotometer (UV 1800 model) was used to analyze turbidity, UV 254 , and UV 260 . Shimadzu atomic absorption spectrophotometer (AA 6300 model) was used to determine concentrations of residual aluminum in water samples and coagulated water.

Results and Discussion
Several factors were analyzed after finishing jar-tests to find coagulation efficiencies for each state of algae cell and followings are the results. For controls, a rapid turbidity removal was observed with the first jar-test, 1 Al-mg coagulant dose and the additional removal was shown with the right next jar-test, 2 Al-mg coagulant dose. Turbidity removals for other jar-tests, over 3 Al-mg coagulant dose, were slightly better than that for the second jar-test, 2 Al-mg coagulant dose and stayed in similar levels. Patterns of turbidity removal for broken cell samples were similar to those for control. The results indicate that coagulation process may not inhibit if algae cells are not existed. Jar-tests over 3 Al-mg coagulant dose for pure cell samples had similar turbidity removal rates compared to control and broken cell samples, but jar-tests with 1 and 2 Al-mg coagulant dose for pure cell samples showed worse turbidity removal rates. Pure cells, therefore, may inhibit coagulation processes more than organic matters from Microcystis aeruginosa do.

Turbidity in Jar-Tested Water
Lastly, turbidities for coagulated water of untreated cell samples showed higher levels than those of other samples although coagulant doses were increased which results showed that coagulation inhibitions were greater when cells and organic matters from Microcystis aeruginosa coexist rather than cells or organic matters are present singly. The similar result could be found in the previous study which reported that co-existence of algae cells and algae originated matter (AOM) may cause of decreasing coagulation efficiency; 95% of algae cells can be removed by coagulation process while AOMs may not be removed [24]. Treating, therefore, either algae cells or organic matters released from algae might significantly reduce coagulation inhibitions when dealing with water resources during waterbloom.

Residual Aluminum in Jar-Tested Water
To check the absorption capability of coagulant towards Microcystis aeruginosa cell itself or organic matters, residual aluminum were measured after jar-tests and Fig. 2 shows concentrations of residual aluminum in coagulated water for all samples. Residual aluminum concentrations of coagulated water for control samples showed the lowest levels and those for pure cell samples did the highest as the previous study showed that the increase in algae count and turbidity causes the rise of the concentrations of residual alumimum [25]. The results are considered to be due to the fact that algae cell sizes are much bigger than those of AOMs or kaolin particles. In other words, surface areas of pure cells which could contact coagulants are smaller than those of organic matters or kaolin particles, so that pure cell samples require less coagulants than other samples. Additionally, kaolin particles agglomerate around large algae cells to form larger flocs to settle down which also makes less coagulant consumptions.
Another issue is that residual aluminum concentrations for jar-tests over 3 Al-mg coagulant dose remained at similar levels and same patterns were shown for turbidity reductions. The 3 Al-mg/500 mL of coagulant, therefore, is the optimum dose for turbidity removal and residual aluminum.

E 254 and E 260 of Jar-Tested Water
E 254 and E 260 indicate UV 254 and UV 260 related substance, respectively. Specific UV wavelengths, UV 254 and UV 260 , are used to determine the amount of organic matter in water. E 254 related to UV 254 indicates aromatic compounds is an indirect indicator for Trihalomethans (THMs, one of carcinogens) precursors and E 260 related to UV 260 indicates for biodegradable organic matters is an indirect indicator for persistent organic pollutants (POPs) in the water [26][27][28].
The study focuses on E 254 and E 260 since byproducts of Microcystis aeruginosa have been known to contain these organic matters [2]. Algae cells release the most abundant E 254 and E 260 during their log-growth phase and stationary phase rather than their lag phase and death phase and this is another reason why the study focused on cells in log-growth phase and stationary phase since they may affect most on drinking water treatment systems [29].  As mentioned above, AOMs may not be removed although algae cell itself could be eliminated by coagulation process [24]. Pre-treatment such as photocatalytic before coagulation process could increase the removal efficiency for AOMs [30]. Fig. 3 and Fig. 4 show changes of E 254 and E 260 in jar-tested water. Changes of E 254 and E 260 in jar-tested water for all samples had comparable patterns to each other. It is obvious that samples with organic matters -untreated cells and broken cells -showed higher levels of E 254 and E 260 after jar-tests than pure cell samples did. Levels of E 254 and E 260 for pure cell samples were even lower than those for controls. As results, SOMs may not related to E 254 and E 260 because pure cell samples only have SOMs while others have IOMs and/or EOMs including SOMs. In addition, E 254 and E 260 , precursors of THMs and POPs, are more in EOMs than IOMs since both untreated cells and broken cells have EOMs.

Conclusions
The study examined coagulation characteristics for different cell states of Microcystis aeruginosa, most common species during water blooms, to give better ideas for researchers, managers and operators who focused on drinking water treatment facilities when waterbloom occurs on their water resources. Coagulation characteristics of Microcystis aeruginosa revealed different behaviors in each cell state.
For the single existence of pure cells or organic matters, broken cell samples showed better turbidity removal rates than pure cell samples did, so pure cells may inhibit coagulation processes more than organic matters from Microcystis aeruginosa do. Turbidities for coagulated water of untreated cell samples showed higher levels than those of other samples in all jar-tests with different coagulant doses which results showed that coagulation inhibitions were greater when cells and organic matters coexist rather than cells or organic matters are present individually. Treating, therefore, only one of algae cells or organic matters released from algae might significantly reduce coagulation inhibitions when waterbloom occurs in water resources.
Pure cell samples showed the highest concentrations for residual aluminum after jar-test. Facts that algae cells are bigger than algae-derived organisms or kaolin particles and kaolin particles agglomerate around large algae cells to form larger flocs to settle down makes less coagulant consumptions.
Also, residual aluminum concentrations after jar-tests were inversely proportional to turbidity removal rates, but their patterns were analogous; their concentrations remained in similar levels for jar-tests over 3 Al-mg coagulant dose. The optimum coagulant dose, therefore, for turbidity removal is 3 Al-mg/500mL, so the coagulant dose should exceed its critical point to obtain better removal rates, but should not exceed it by too much for the sake of cost efficiency.
The removal patterns for E 254 and E 260 of all cell states samples were similar. Samples with organic matters showed higher E 254 and E 260 and levels of E 254 and E 260 for pure cell samples were even lower than those for controls. SOMs, therefore, may not related to E 254 and E 260 and EOMs may be the main sources of E 254 and E 260 since both untreated cells and broken cells have EOMs. The results indicate that water treatment facilities should pay special attentions to EOMs, so that they could remove potential risks for public health from Microcystis aeruginosa and its byproducts. Moreover, it is better to remove organic matters rather than algae cell itself in order to reduce the coagulation inhibition since organic matters could have potential risks to the public health.
The study will assist water treatment facilities in developing specific techniques or processes during waterbloom to reduce potential risks for public health as well as water treatment process inhibitions from cyanobacteria and its byproducts.