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 . 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 . 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 . In a line, Azov and Goldman  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.  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.  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 . 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 :
The experiment was conducted for a maximum of 8 days contact. The specific rate of NH4-N removal (Rs) was also estimated, using the known Eq. (2):
where (Chl a)o stands for the initial concentration of Chl a at the time to. 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 .
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 . 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 . Martinez et al.  reported over 97% nitrogen removal by Scenedesmus obliquus for the initial concentration of 27.4 mg N/L. Olguin  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. . Nevertheless, few reports showed higher or more efficient NH4-N removal, even at higher concentrations of nitrogen. Shi et al.  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  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 . 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 . However, the lower removal rate was reported as 3.4 mg N/L/day of Chlorella pyrenoidosa . Akpor and Muchie  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.  reported a substantially higher removal rate of 83 mg N/L/day, upon using the suspended growth culture Scenedesmus sp. Similarly, Wang and Lan  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  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.