**2. Experimental**

#### **2.1 Reagent and algae cell culture**

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.

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

Concentration of phosphate ion (PO4-P) was determined by the molybdenum blue method (JIS K-0102). Inorganic nitrogen (inorganic-N) value was calculated from sum the of NH4 + , NO2 <sup>−</sup>, and NO3 <sup>−</sup> concentrations. The pH value was measured using 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).

## **3. Results and discussion**

#### **3.1 Backgrounds of the present study**

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

**27**

**Figure 5.**

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

using biosensors [2, 42] and remediation techniques for closed water quality [43].

The pond investigated in this time is on the site of the university and is an

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

, an average depth of ca. 1 m, a

The present study was carried out based on these backgrounds.

water quality necessary for the small aquatic organisms to survive.

*Photograph of thick-layered water bloom on the surface water of the pond (2006).*

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

**3.3 Periodic monitoring of water quality**

a depth of about 10 cm at 10:30 am.

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

**3.2 Investigation of aquatic ecosystem**

adjustment reservoir with an area of 20,000 m<sup>2</sup>

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

using biosensors [2, 42] and remediation techniques for closed water quality [43]. The present study was carried out based on these backgrounds.
