**3.2 Investigation of aquatic ecosystem**

*Water Chemistry*

**2. Experimental**

sum the of NH4

+ , NO2

**3. Results and discussion**

**3.1 Backgrounds of the present study**

**2.1 Reagent and algae cell culture**

blue-green algae, iron salt, aluminum salt, permanganate potassium composite, or calcium phosphate precipitation was employed as a coagulation reagent [31–34]. In the calcium phosphate precipitation method, phosphate ion was intensively precipitated with calcium ion by addition of sodium hydroxide [34]. Blue-green algae are coagulated by calcium phosphate precipitation due to that the algae cell surface is negatively charged [35, 36]. The coagulate substance is suitable for algae biomass

As described above, most of chemical remediation methods have problems as the environmentally friendly method. In the present study, we propose a water chemical remediation (WCR) system for simultaneous removal of phosphate ion and blue-green algae from the surface water of the anthropogenically eutrophied pond [37]. The system employs calcium chloride dihydrate for both precipitation with phosphate ion and coagulation with blue-green algae (**Figure 4**). The calcium chloride dihydrate has no ecotoxicity, low toxicity reagent (LD50 2045 mg/kg, rat male/oral), and widely used as food additive and snow-melting agent in Japan. Further, calcium chloride has high water solubility (74.5 g/100 mL at 20°C); the feature is able to reduce the amount of the reagent. For the study, we had been investigating the water quality of the anthropogenically eutrophied pond from

Calcium chloride dihydrate was purchased from Wako Chemicals, and the other chemicals were used for analytical grade. The growth of *M. aeruginosa* NIES-87 as a model of blue-green algae: *M. aeruginosa* NIES-87 was purchased from the National Institute for Environmental Studies and grown by two-step incubations. As preincubation, *M. aeruginosa* was grown in a test tube with 10 mL of MA medium until reaching stationary phase (120 rpm, 27°C, light-dark cycle of 12 h) [39]. As present incubation, the growth medium of the preincubation was added to a glass incubation bottle containing 700 mL of MA medium and grown under 27°C for 2 weeks.

Concentration of phosphate ion (PO4-P) was determined by the molybdenum blue method (JIS K-0102). Inorganic nitrogen (inorganic-N) value was calculated from

a pH electrode (model, IOL-50, DKK, Japan). Dissolved oxygen (DO) was measured using a DO meter (model, MM-60R, TOA-DKK, Japan). Chemical oxygen demand (COD) was measured by a potassium permanganate method. Total organic carbon (TOC) was measured using a TOC meter (model, TOC-5000A, Simadzu, Japan).

When the author was a doctoral student, the author studied on enzyme biosensors for phosphate ion in the reserved water for drinking [40, 41]. Then after, the author had interests to the phenomenon of anthropogenic eutrophication [38] and subsequent organic pollution [1] and also had interests to environmental protection

<sup>−</sup> concentrations. The pH value was measured using

production due to contain calcium phosphate precipitation as nutrient salt.

2006 to 2009 [38]. The results are also shown in the present study.

**2.2 Instruments and methods for water quality monitoring**

<sup>−</sup>, and NO3

**26**

The pond investigated in this time is on the site of the university and is an adjustment reservoir with an area of 20,000 m<sup>2</sup> , an average depth of ca. 1 m, a maximum depth of ca. 3 m, and a storage capacity of about 20,000 kL. Most of the pond water is the inflow of rainwater that has fallen into the university. In addition, the surplus of the treated sewage that is not used for the regeneration of toilet flushing water is discharged to the pond as drainage. In addition, the pond bottom is covered with a rubber sheet to prevent the penetration of the pond water [38].

In this pond, water blooms were often observed by the rise of the water temperature (**Figure 2d**). In 2006, the thick layer of the water bloom on the surface water had been seen at the corner of the pond (**Figure 5**). Using a microscope, two types of blue-green algae in the form of spherical *Microcystis* sp. and filamentous *Planktothrix* sp. were observed from the surface water. Subsequently, the ecology of aquatic animals was investigated. In the case of hypertrophic water body, DO is consumed by aerobic respiration of blue-green algae at night, and then the water body changes to reductive environment [14]. In such reductive condition, survival of small fishes becomes more difficult than that of large fishes. In fact, only large carps and large turtles were observed by visual observation of the water surface. Then, we tried to capture small aquatic organisms such as small fishes using a cell bottle, a four-way net, and casting net. As a result, small fishes such as *Cyprinus carpio* and *Carassius* were observed. Therefore, it was found that the pond maintained the water quality necessary for the small aquatic organisms to survive.

#### **3.3 Periodic monitoring of water quality**

Periodic monitoring was conducted twice a month to examine several items related to weather conditions, basic properties of water quality, eutrophication, and organic pollution. Water sampling was conducted by collecting reservoir water with a depth of about 10 cm at 10:30 am.

**Figure 6.**

*Periodic monitoring for indications of basic water quality (2008) [38]. \* The Ministry of the Environment (Japan), water-pollution standard, Appendix 2.*

**Figure 7.**

*Periodic monitoring for indications of eutrophication (fiscal years between 2006 and 2009) [38]. a) Phosphate ion and b) inorganic nitrogen. \* The Ministry of the Environment (Japan), water-pollution standard, Appendix 2.*

In the indications of the basic water quality, the values of both pH and DO are influenced by the intensive photosynthesis of blue-green algae (**Figure 6**). The annual average values of both pH and DO were pH 9.2 ± 0.32 and 14.2 ± 4.4 mg O2/L, respectively. The pH value was above the environmental standard, and the DO value indicated that it was supersaturated. It was speculated that these water quality conditions were caused by intensive photosynthesis of the blue-green algae [13].

In the indications of eutrophication, the concentrations of phosphate ion (PO4-P) and inorganic nitrogen (inorganic-N) exceeded each environmental standard value in most cases (**Figure 7**). In general, intensive growth of blue-green algae consumes these nutrient salts [44]. Nevertheless, such reduction of nutrient salt concentrations was not observed in this pond. The results supported that the treated sewage flowing into this pond always supplies these nutrients.

**29**

**Figure 9.**

*Water Chemical Remediation for Simultaneous Removal of Phosphate Ion and Blue-Green Algae…*

In the indications of organic pollution, the values of COD and TOC did not exceed each environmental standard value in most cases (**Figure 8**). The results showed that the organic substances did not suspend so much in the surface water, although measurement sample was filtrated using 0.45 μm filter as pretreatment. However, it was found that sludge had been accumulated on the bottom of the pond

*The Ministry of the Environment* 

A coagulation test was performed using growth medium of *M. aeruginosa* NIES-87. CaCl2 solution (0.1 M) of 250 mL and was added to 600 mL of the growth medium, and distilled water was finally added to become 1 L in a measuring

*Constitution of a sand filter for pre-examination. (a) Growth medium of M. aeruginosa NIES-87, (b) coagulation of M. aeruginosa using CaCl2, (c) enlarged view of the coagulation, (d) micrograph of* 

*M. aeruginosa coagulated (×20), (e) M. aeruginosa filtered, and (f) filtrate.*

**3.4 Coagulation test and simultaneous removal tests**

*Periodic monitoring for indications of organic pollution (2008) [38]. \**

*(Japan); water-pollution standard, Appendix 2.*

*DOI: http://dx.doi.org/10.5772/intechopen.88490*

(data not shown).

**Figure 8.**

*Water Chemical Remediation for Simultaneous Removal of Phosphate Ion and Blue-Green Algae… DOI: http://dx.doi.org/10.5772/intechopen.88490*

**Figure 8.**

*Water Chemistry*

**Figure 6.**

*Periodic monitoring for indications of basic water quality (2008) [38]. \**

*(Japan), water-pollution standard, Appendix 2.*

**28**

**Figure 7.**

*Appendix 2.*

blue-green algae [13].

*ion and b) inorganic nitrogen. \**

In the indications of the basic water quality, the values of both pH and DO are influenced by the intensive photosynthesis of blue-green algae (**Figure 6**).

*Periodic monitoring for indications of eutrophication (fiscal years between 2006 and 2009) [38]. a) Phosphate* 

*The Ministry of the Environment (Japan), water-pollution standard,* 

*The Ministry of the Environment* 

In the indications of eutrophication, the concentrations of phosphate ion (PO4-P) and inorganic nitrogen (inorganic-N) exceeded each environmental standard value in most cases (**Figure 7**). In general, intensive growth of blue-green algae consumes these nutrient salts [44]. Nevertheless, such reduction of nutrient salt concentrations was not observed in this pond. The results supported that the

The annual average values of both pH and DO were pH 9.2 ± 0.32 and 14.2 ± 4.4 mg O2/L, respectively. The pH value was above the environmental standard, and the DO value indicated that it was supersaturated. It was speculated that these water quality conditions were caused by intensive photosynthesis of the

treated sewage flowing into this pond always supplies these nutrients.

*Periodic monitoring for indications of organic pollution (2008) [38]. \* The Ministry of the Environment (Japan); water-pollution standard, Appendix 2.*

In the indications of organic pollution, the values of COD and TOC did not exceed each environmental standard value in most cases (**Figure 8**). The results showed that the organic substances did not suspend so much in the surface water, although measurement sample was filtrated using 0.45 μm filter as pretreatment. However, it was found that sludge had been accumulated on the bottom of the pond (data not shown).

## **3.4 Coagulation test and simultaneous removal tests**

A coagulation test was performed using growth medium of *M. aeruginosa* NIES-87. CaCl2 solution (0.1 M) of 250 mL and was added to 600 mL of the growth medium, and distilled water was finally added to become 1 L in a measuring

#### **Figure 9.**

*Constitution of a sand filter for pre-examination. (a) Growth medium of M. aeruginosa NIES-87, (b) coagulation of M. aeruginosa using CaCl2, (c) enlarged view of the coagulation, (d) micrograph of M. aeruginosa coagulated (×20), (e) M. aeruginosa filtered, and (f) filtrate.*

cylinder (P:Ca = 1:100 in molar ratio). Then, *M. aeruginosa* was coagulated immediately (**Figure 9**). After leaving for half a day, the medium was used for subsequent simultaneous removal tests.

A medium of 100 mL flowed into a sand filtration column which was prepared for three columns (**Figure 10**). The column was designed for reverse cleaning to recover both phosphate ion and *M. aeruginosa* from the sample. The results showed the possibility as a simultaneous removal of phosphate ion and *M. aeruginosa* (**Table 1**). Next, we examined using the pond water in the same way. As a result, phosphate ion in the pond water (adjusted to pH 10.5) could be removed 65.8% from 0.719 to 0.246 ± 0.0023 mg/L (*n* = 3). Based on these results, we next constructed a simultaneous removal system.
