Nitrogen is a one of the most vital nutrients for aquatic plants and algae. However, an excessive concentration of nutrients (containing nitrogen) will stimulate aquatic plant and algal growth and will cause serious pollution problems. In order to prevent these problems, we should handle the nitrogen as a nutrient resource rather than a pollutant that only has to be disposed off. Biological treatment using microalgae is one of the potential treatments to reduce the nitrogen, where the nitrogen is used as a nutrient for the microorganisms.

Many algal species, especially the family of Chlorella genus, are found to be tolerant to organic pollutants and could rapidly colonize the given nutrients, such as nitrogen, phosphorus, and organic compounds [1]. The advantages of using algae for this purpose include low operational costs; the possibility of recycling assimilated nitrogen and phosphorus into algae biomass as a fertilizer, avoiding sludge handling problems; and finally, the discharge of oxygenated effluent into water bodies. Moreover, this process is not associated with carbon, as is usually required for nitrogen and phosphorus removal, which is an additional advantage for the treatment of secondary effluents. Microalgae require nitrogen, phosphorus, CO₂, and light for autotrophic growth [2]. Additionally, microalgae are very sensitive to the combined effect of high NH₄-N concentrations and high pH values, because NH₄-N uncouples the electron transport in photo system II and competes with H₂O in the oxidation reactions leading to O₂ generation [3]. In a line, Azov and Goldman [4] observed a significant decrease in the efficiency at high pH (i.e., pH 8) and NH₃ concentrations (i.e., 2 mM) in an algae-containing pond. Similarly, Munoz et al. [5] reported a complete inhibition of Chlorella sorokiniana at an NH₃/NH₄+ concentration of 15 mM at pH 8.7, during the photosynthetically oxygenated treatment of 2 g/L of acetonitrile, in a 50-L column photobioreactor. However, effective use of NH₃-tolerant microalgae can improve the stability of this process. Ogbonna et al. [6] reported no significant effect on the growth of C. sorokiniana, even at 22 mM NH₃, whereas Spirulina platensis was almost inhibited at very low concentrations of NH₃ (i.e., 11 mM).

One limitation in employing an algal system as the secondary treatment process is the presence of high concentrations of ammonia and urea in raw wastes, especially those discharged from the livestock and food industries, which inhibit algae growth and physiological activity [7]. However, it has been noted that the studies undertaken previously were mainly focused on the effects of N-deficiency, and competitive interaction between nitrate and ammonia uptake at low N level. Relatively little information is available on ammonia removal using different ammonia concentrations in real wastewater treatment by Chlorella vulgaris (C. vulgaris).

Based on the aforementioned reasons, in the present investigation we have aimed to explore and examine the efficiency of C. vulgaris in the removal of ammonia-nitrogen from wastewaters containing varied concentrations of ammonia-nitrogen.

Cells of C. vulgaris (FC-16) were cultured in Jaworski’s Medium in deionized water, with LED lamps at an ambient temperature. Jaworski’s Medium is composed of 4.0 g Ca(NO₃)₂·H₂O, 2.48 g KH₂PO₄, 10.0 g MgSO₄·7H₂O, 3.18 g NaHCO₃, 0.45 g EDTAFeNa, 0.45 g EDTANa₂, 0.496 g H₃BO, 0.278 g MnCl₂·4H₂O, 0.20 g (NH₄)6Mo7O₂₄·4H₂O, 0.008 g Cyanocobalamin, 0.008 g thiamine HCl, 0.008 g biotin, 16.0 g NaNO₃, and 7.2 g Na₂HPO₄·12H₂O per 200 mL. The cultures were incubated at a constant temperature of 25℃ ± 2℃ and continuous light intensity of 100 µE/m²/s for 15 days. Cultures of C. vulgaris (FC-16) ranged in size from 3–8 μm and were round in shape.

The raw wastewater was obtained from the preliminary sedimentation of a sewage plant at Gangneung, Korea. Table 1 shows the characteristics of the wastewater used throughout our investigation.

The analyzed raw wastewater was noted to be favorable for treatment with microalgae and removal of the available nutrients. An excess ratio of chemical oxygen demand, nitrogen, and phosphorus (i.e., 100:20:2) for this wastewater is recommended for nutrient removal in activated sludge plants. The biochemical oxygen demand (BOD₅) and total phosphorus (TP) and BOD₅ and PO₄ ratios were found to be reasonably high. Similarly, the PO₄ and TP ratio was at its higher range for municipal wastewater. Lastly, calcium, potassium and manganese were not limiting for biological wastewater treatment, and iron was naturally present in the wastewater.

To eliminate bacteria and protozoa, the wastewater samples were sterilized by autoclaving for 30 min. The experiments were conducted using a batch reactor operation with 1 L conical flasks. At the beginning of each series of experiments, 500 mL of wastewater was inoculated to the flasks with pre-cultured C. vulgaris. To evaluate the efficiency of C. vulgaris in the removal of ammonia-nitrogen from wastewaters, eight different fractions were prepared with varied concentrations of ammonia-nitrogen. The experimental design adopted is represented in Table 2.

The initial cell density was 1 × 10⁶ cells per milliliter for each experimental set up. The initial chlorophyll a (Chl a) concentration was kept constant at 2.5 ± 0.5 mg/L, throughout the experiments. The NH₄-N concentration was varied from 15.22 to 205.29 mg/L. The entire experiment was conducted at a neutral pH (7.5 ± 0.3), constant temperature of 25℃ ± 1℃, a light intensity of 100 µE/m²/s, and a dark-light cycle of 8–16 hr for 8 days. Raw wastewater was used for this experiment.

The pH values of the cultures were measured intermittently and maintained a constant value of pH (7.5 ± 0.3) by the addition of sterilized and diluted NaOH or HCl.

Dry-weight estimations do not exclusively monitor the amount of algae, because bacteria and zooplankton may add to the biomass. Because only algae contain chlorophyll, the estimation of this pigment was a reliable though elaborate method in algae biomass computation. Depending on the algal strain examined, acetone, ethanol, or diethyl ether was used to extract the pigment from the separated algal cells. In some cases, brief heating was required to achieve complete pigment extraction. After that, the cell debris was removed by centrifugation or filtration, and the extract was protected from light to avoid bleaching of the pigments.

The Chl a concentration in the extract was calculated, by reading the absorbance (A) of the pigment extract in a spectrophotometer at a given wavelength against a solvent blank by using Eq. (1) as follows [8]:

The experiment was conducted for a maximum of 8 days contact. The specific rate of NH₄-N removal (R_s) was also estimated, using the known Eq. (2):

where (Chl a)_o stands for the initial concentration of Chl a at the time t_o. A specific form of Chl a was used in oxygenic photosynthesis.

NH₄-N concentration was measured by ion chromatograph (Metrohm AG, Herisau Switzerland), and the analysis was performed according to the standard methods described elsewhere [9].

The variation of NH₄-N concentration as a function of time with various initial NH₄-N concentrations is depicted in Fig. 1.

The maximum NH₄-N removal efficiency was obtained after 8 days, and the values were found to be 99.61%, 99.52%, 72.32%, 61.32%, 50.22%, 31.31%, 22.99%, and 3.59% for Runs 1–8, respectively. NH₄-N was completely removed by C. vulgaris, when the initial concentration was between 15.22–25.24 mg/L. However, the NH₄-N removal was approximately 50% at an NH₄-N concentration of 85.52 mg/L, and the NH₄-N levels further decreased to less than 32% at the NH₄-N concentration taken beyond 105.43 mg/L. Only 3.59% of the removal efficiency was obtained for the 200 mg/L concentration of NH₄-N. Further, it was observed that the NH₄-N removal efficiency obtained after 2 days was 93.69% for Run 1, 78.49% for Run 2, 41.94% for Run 3, 37.36% for Run 4, 35.23% for Run 5, 24.56% for Run 6, 16.02% for Run 7, and 3.04% for Run 8. The reported NH₄-N removal efficiencies varied, depending on the media composition and environmental conditions, such as the initial nutrient concentrations, light intensity, light/dark cycle, and algae species [10]. The NH₄-N removal efficiency achieved in this study was higher, compared to that of other studies; an average of 72% nitrogen removal was reported for C. vulgaris from 3–8 mg NH₄-N/L containing diluted ethanol and citric acid production effluent [11]. Martinez et al. [12] reported over 97% nitrogen removal by Scenedesmus obliquus for the initial concentration of 27.4 mg N/L. Olguin [13] obtained a maximum of 96% NH₄-N removal by Spirulina in an outdoor raceway as a result of treatment with 2% diluted anaerobic effluents from pig wastewater containing almost the same amount of nitrogen as in the experiment carried out by Martinez et al. [12]. Nevertheless, few reports showed higher or more efficient NH₄-N removal, even at higher concentrations of nitrogen. Shi et al. [14] investigated the effect of the initial nitrogen and phosphorus concentrations on the nutrient removal performance of the algae Botryococcus braunii from secondary treated piggery wastewater. The culture was able to consume the available NO₃-N completely, i.e., up to 510 mg/L within 6 days of batch operation. Aslan and Kapdan [2] investigated the batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by C. vulgaris. This study showed that a 21.2 mg/L concentration of NH₄-N was removed, using the microalgae C. vulgaris.

Nitrogen is the major constituent of proteins, chlorophyll, and enzymes involved in photosynthesis. Therefore, nitrogen affects the photosynthesis of microalgae. The nitrogen absorbed by C. vulgaris mostly includes NO₃-N and NH₄-N, and their uptake, deposition, and assimilation in C. vulgaris are different. Chlorophyll is an extremely important biomolecule that is critical in photosynthesis, and that allows plants to absorb energy from light. The function of the vast majority of chlorophyll (up to several hundred molecules per photosystem) is to absorb light and transfer that light energy by resonance energy transfer to a specific chlorophyll pair in the reaction center of the photosystem [15]. Chl a (C₅₅H₇₂O₅N₄Mg) is important in the energy phase of photosynthesis. Two electrons are needed for the electron acceptors to proceed in the photosynthesis process. Within the reaction centers of both photosystems is a pair of Chl a molecules that transfer electrons to the transport chain through redox reactions. Chl a is a common pigment found in algae. This pigment is what algae use to trap energy from light to promote algal growth. The total Chl a content (mg/L) was obtained and is represented in Fig. 2 for the wastewater samples. It was noted that the total Chl a content increased gradually with the incubation time in all cultures, with the highest Chl a content recorded at day 15 (data not presented).

Fig. 2 clearly demonstrates that the final Chl a content of the culture significantly increased from Run 1 to Run 4. However, it decreased gradually beyond Run 5, exceeding 85.52 mg/L of total NH₄-N (Fig. 2). This result suggests that at low NH₄-N concentrations, Chl a formation was limited by NH₄-N supply, while excessive NH₄-N concentration does not favor the additional synthesis of Chl a. The maximal value of Chl a content was 19.21 mg/L for Run 4. These results indicate that C. vulgaris is effective in removing NH₄-N concentration at Run 4 (minimal removal efficiency of 60% NH₄-N). In this study, the removal efficiency of NH₄-N up to Run 5 was less than 50%.

The batch data was further utilized to discuss the kinetics of NH₄-N removal. The initial NH₄-N removal rate was used to determine the coefficients. The removal rate (R) was calculated for these wastewater samples, and the obtained values are represented in Table 3.

The maximum NH₄-N removal rate was found to be 5.37 mg/L/day for Run 4. Run 4 sample was obtained 2.8 times and 5.8 times higher than that of Run 1 and Run 8, respectively. The

NH₄-N removal rates were similar to those obtained previously by other related studies. The nitrogen removal rate by C. vulgaris was reported to be 5.44 mg/L/day [15]. However, the lower removal rate was reported as 3.4 mg N/L/day of Chlorella pyrenoidosa [16]. Akpor and Muchie [17] also reported somewhat lower removal rates (i.e., 3.36–3.60 mg NH₄-N/L/day, 1.2–3.6 mg NH₄-N/L/day, and 1.2–3.12 mg NH₄-N/L/day by Aspidisca, Trachelophyllum and Peranema, respectively). In contrast, Park et al. [18] reported a substantially higher removal rate of 83 mg N/L/day, upon using the suspended growth culture Scenedesmus sp. Similarly, Wang and Lan [19] reported maximal removal rate of 43.7 mg/L/day at 140 mg N/L for Neochloris oleoabundans.

The initial cell density was 1 × 10⁶ cells per milliliter, and the initial Chl a concentration was kept constant at 2.5 ± 0.5 mg/L throughout the experiments. The specific NH₄-N removal rate for varying NH₄-N concentration was calculated and is depicted in Fig. 3.

NH₄-N absorbed by C. vulgaris can be directly used, but the absorbed NO₃-N cannot be used until it is deoxidized to NH₄-N, and the processes consume energy and reducing power. Therefore, NH₄-N can be utilized rapidly at the early stage, which is in favor of chloroplast synthesis and can promote photochemical efficiency. The maximum specific NH₄-N removal rate was found to be 1.79 mg/mg Chl a/day for Run 5. At higher concentrations of NH₄-N (after Run 5), the removal rates decreased gradually. These results indicated that the physiological activity of C. vulgaris was reduced by high NH₄-N concentrations. The first is that excessive NH₄-N can damage photosynthesis organs and decrease photochemical efficiency, the other is that excessive NH₄-N can markedly increase the ability of chloroplasts to

dissipate the excessive energy. So they cannot efficiently utilize the photon energy absorbed by pigments for photosynthesis. NH₄-N partly replacing NO₃-N decreases the consumption of energy and reducing power, while NO₃-N partly replacing NH₄-N relieves metabolic disorder induced by the excessive NH₄-N, and makes the physiological metabolism in C. vulgaris. The NH₄-N removal rates, rather than specific removal rates, were reported in most of the previous studies. Aslan and Kapdan [2] reported the specific NH₄-N removal rate from 0.6 to approximately 0.9 mg/mg Chl a/day for the NH₄-N concentrations between 60 to 125 mg by C. vulgaris.

In this study, the potential of C. vulgaris for the removal of various concentrations of ammonia-nitrogen from wastewaters using batch reactor operations was evaluated. From the results, it was found that NH₄-N was completely removed by C. vulgaris in the initial concentration range 5.22–25.24 mg/L. Therefore, C. vulgaris is more suitable for domestic wastewater treatment, than that of industrial wastewater treatment containing NH₄-N. The maximal value of Chl a content was found to be 19.21 mg/L for 65.79 mg/L NH₄-N concentration. However, the maximum specific NH₄-N removal rate was found to be 1.79 mg/mg Chl a/day, with the initial NH₄-N concentration of 85.52 mg/L. At higher concentrations of NH₄-N (after Run 5), the removal rates gradually decreased. From the results, it can be concluded that the physiological activity of C. vulgaris was reduced by high NH₄-N concentrations. The plausible reason for this is that first, excessive NH₄-N can damage photosynthesis organs and decrease photochemical efficiency. Further, excessive NH₄-N can markedly increase the ability of chloroplasts to dissipate the excessive energy.