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Journal of Environmental Biology
ISSN: 0254- 8704 CODEN: JEBIDP

Nitrogen and phosphorus removal from municipal wastewater by the green alga Chlorella sp.
Changfu Wang, Xiaoqing Yu, Hong Lv and Jun Yang*
Aquatic Ecohealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, People's Republic of China *Corresponding Author email : jyang@iue.ac.cn

Abstract
The potential of microalgae as a source of renewable energy based on wastewater has received increasing interest worldwide in recent decades. A freshwater microalga Chlorella sp. was investigated for its ability to remove both nitrogen and phosphorus from influent and effluent wastewaters which were diluted in four different proportions (namely, 100%, 75%, 50% and 25%). Chlorella sp. grew fastest under 50% influent and effluent wastewaters culture conditions, and showed an maximum cell density (4.25109 ind l1 for influent wastewater and 3.54109 ind l1 for effluent wastewater), indicating the levels of nitrogen and phosphorus greatly influenced algal growth. High removal efficiency for total nitrogen (17.0458.85%) and total phosphorus (62.43 97.08%) was achieved. Further, more than 83% NH 4N in 75%, 50%, 25% influent wastewater, 88% NOXN in effluent wastewater and 90% PO 4P in all treatments were eliminated after 24 days of incubation. Chlorella sp. grew well when PO4 P concentration was very low, indicating that this might be not the limiting factor to algal growth. Our results suggest the potential importance of integrating nutrient removal from wastewater by microalgae cultivation as biofuel production feedstock.

Publication Info
Paper received: 28 May 2012 Revised received: 21 September 2012 Accepted: 17 December 2012

Key words
Microalgae, Municipal wastewater, Nutrient removal, Biofuel

Introduction Human activities, particularly agriculture and urbanization, have led to increased nitrogen and phosphorus discharge to inland water systems. This nutrient enrichment or eutrophication can profoundly alter the structure and function of aquatic ecosystems, potentially endangering human health, biodiversity and ecosystem sustainability. Therefore, both nitrogen and phosphorus in wastewater should be properly treated or reused thereby reducing their contaminant effects in aquatic ecosystems (An et al., 2003). In the past special attention has been focused on nitrogen and phosphorus removal from municipal wastewater using biological, physical and chemical methods (Blackall et al., 2002; Mallick, 2002). However, some harmful substances can not be effectively eliminated because the conventional treatment technology used in wastewater
Triveni Enterprises, Lucknow (India )

treatment plants is insufficient for removing these specific compounds (Ternes, 1998; Saan and Balcioglu, 2006). More often, the effluents from the wastewater treatment plant fail to meet with the national or local environmental standards. Recent studies have demonstrated that microalgae have a great potential for the removal of nitrogen and phosphorus from wastewater (An et al., 2003; Blackall et al., 2002; Mallick, 2002; rpeza et al., 2009). Microalgae can be used for treatment of wastewater due to their capacity to assimilate nutrients including both nitrogen and phosphorus (Noe et al., 1992; Shi et al., 2007). The advantages of using microalgae for that purpose include: the possibility of recycling assimilated nitrogen and phosphorus into algae biomass as a fertilizer, the low cost of the operation as these inorganic nutrients in wastewaters are suitable and costeffective for microalgae cultivation, the reduction of the life-cycle freshwater usage by as much as 90%, and a lack
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Table 1 : Physico-chemical parameters of wastewater Influent pH Total nitrogen NO x-N NH 4 -N Total phosphorus PO4 -P Zinc Copper Cadmium Chromium Lead 7.5 38.03 0.75 16.30 1.83 0.64 <0.030 0.065 0.34 <0.036 0.60 Effluent 7.5 29.45 24.4 1.13 0.57 0.52 <0.030 0.055 0.28 <0.036 0.24

of competition with existing food production on land (Gonzlez et al., 1997; Li et al., 2010; Yang et al., 2011). Moreover, microalgae cultures offer an interesting alternative for wastewater treatment because they provide a biotreatment coupled with the production of potentially valuable biomass with implications for the reduction of greenhouse gas emission (Sawayama et al., 1999; Kim et al., 2010; Pittman et al., 2011). Microalgae treatment of wastewater does not generate additional pollution thus it can offer an ecologically safer, cheaper and more efficient means of removing nutrients than conventional methods (Clarens et al., 2010; Wijffels and Barbosa, 2010). Globally Chlorella is one of the most extensively used microalgae for nutrient removal. It is a unicellular freeliving green alga which is widely distributed around the world. Most species of this genus are spherical and under 10 m in diameter. More importantly, Chlorella has become a good candidate for biofuel production due to its rapid growth rate and high lipid content (Bashan et al., 2002; Xu et al., 2006; Li et al., 2007). The use of Chlorella for wastewater treatment is not a new idea, and many researchers have developed techniques for exploiting the fast-growing isolates and increasing their nutrient removal capacity (Aslan and Kapdan, 2006; Xiong et al., 2008). This raises the possibility of the dual-use microalgae cultivation for wastewater treatment coupled with biofuel production. This is an attractive option although so far the application of microalgae in the wastewater industry is still fairly limited. The aim of the present study is to determine the optimum wastewater nutrient concentration for growth of Chlorella sp. and to assess the removal efficiencies of nitrogen and phosphorus from both influent and effluent wastewaters. Materials and Methods Microalga and culture conditions : The microalgae strain (Chlorella sp.) was isolated from Lake Mulan, Central China in July 2010. Pre-cultures were carried out in 150-ml Erlenmeyer flask with light flux density 60 mol photon m-2S-1; light-dark cycle 12hr/12hr; temperature 25 C. Chlorella strain was cultured in a modified Chu 13 medium. Municipal wastewater : Municipal wastewater including influent and effluent was taken from a wastewater treatment plant in Xiamen, China. This plant treated both domestic and industrial wastewaters by the traditional activated sludge method, but the majority wastewater is mainly from domestic sources. The influent and effluent waste wTICAaters were filtered through a 0.22-m pore size membrane. After autoclaving, the wastewaters were moved to 500 ml Erlenmeyer flasks for Chlorella sp. cultivation. The characteristics of waste waters after pre-treatment are

All values are in mgl-1 except for pH

listed in Table 1. The wastewater samples were diluted into four different levels with distilled water, and the proportions of wastewater were 100%, 75%, 50%, 25% for both influent and effluent wastewaters, respectively. Each treatment was conducted in batch by using 500 ml Erlenmeyer flask with three replicates of each treatment. At the beginning of each series of experiments, 300 ml of culture medium was inoculated to flasks. All treatments were in a biological incubator and the cultural conditions were the same as the pre-culture conditions. Microalgal growth monitoring : Growth of Chlorella sp. was determined by cell counts and optical densities were measured at 680 nm using a spectrophotometer (WPA Biowave II UV/Visible Spectrophotometer, UK). At the end of the experiment, cultures were harvested and centrifuged at 5000 rpm for 5 min. The pellets were washed with distilled water and dried at 80 C for 48 hrs before being used to measure dry biomass. Nutrition measurement : TN was determined using a TOCVCPH analyzer (Shimadzu, Japan). TP, ammonium nitrogen (NH4-N), nitrite and nitrate nitrogen (NOx-N) and phosphate phosphorus (PO4-P) were measured with a Flow Injection Analyzer (Lachat Instruments, QC8500, USA). Statistical analysis : All analyses were conducted using the software STATISTICA version 6.0. Results and Discussion Pretreatment of municipal wastewater : The municipal wastewater characteristics are listed in Table 1. In order to eliminate the influence of microorganisms, filtered and sterilized wastewaters were used in this study. Wastewater filtration and sterilization has also been carried out in many previous studies, although it has been shown that these treatments can sometime change the content of nutrients (Sawayama et al., 1992; Gonzlez et al., 1997; rpeza et al., 2009).

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Nutrient removal from wastewater by microalga Table 2 : Total nitrogen content in different proportions of wastewaters removed by Chlorella sp. Influent 75% 28.52 13.830.45 51.511.58 Effluent 75% 22.09 12.710.15 42.462.13

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100% Initial value (mg l ) 38.03 Terminal value (mg l-1) 15.650.59 Removal ratio (%) 58.851.55
-1

50% 19.01 13.790.41 27.462.1

25% ----

100%

50%

25 %

29.45 16.811.20 42.934.07

14.73 -12.220.074 -17.040.50 --

Values are mean of three replicates SE. -- Data not available Table 3 : Total phosphorus content in different proportions of wastewaters removed by Chlorella sp. 100% Initial value (mg l ) 1.83 Terminal value (mg l-1) 0.053 0.0047 Removal ratio(%) 97.08 0.26
-1

Influent 75% 50% 1.37 0.064 0.011 95.35 0.78 0.92 0.065 0.0070 92.91 0.77

25% 0.46 0.045 0.0040 90.12 0.87

100% 0.57 0.046 0.0016 91.98 0.28

Effluent 75% 0.43 0.044 0.0020 89.60 0.47

50% 0.29 0.050 0.0042 82.51 1.50

25% 0.14 0.053 0.0080 62.43 5.62

Values are mean of three replicates SE Algal growth in wastewaters : To explore the optimum culture conditions for Chlorella, the municipal wastewaters were diluted into four different concentrations. The effects of different nitrogen and phosphorus concentrations on algal growth were quite obvious (Fig. 1). Although Chlorella sp. in influent and effluent wastewaters could grow in most culture systems, the highest biomass was obtained in 50% influent wastewater with a maximum cell density 4.2510 9 ind l-1 (dry weight 0.278 g l-1), and in 50% effluent wastewater with the maximum cell density 3.54109 ind l-1 (dry weight 0.258 g l-1) after 24 days of incubation, respectively. However, the slowest growth rates were found in the 100% influent wastewater and 100% effluent wastewater. Thus, the growth of microalgae in wastewater depended on the initial value of the nutrient concentrations. It appeared that there were some inhibitory factors in the initial stage. This phenomenon was also found during the first days of growth of Chlamydomonas reinhardtii in wastewater (Kong et al., 2010). Wastewater often has high concentration of nutrients, much of the N in the form of NH4-N which can inhibit algal growth at high concentration (Wrigley and Toerien, 1990). In addition, the presence of toxic heavy metals and organic compounds in wastewater, especially in industrial wastewater, is another critical inhibition factor for microalgal growth (Chinnasamy et al., 2010). Removal of nitrogen : The Chlorella sp. showed higher removal ratios of total nitrogen (TN) in influent wastewater than effluent wastewater. Interestingly, the higher concentration of wastewater led to the higher removal ratio of TN. Under 100% and 75% wastewater conditions, removal ratios of TN from the influent and effluent wastewaters were higher than 50% and 25 %, respectively (Table 2). The majority of dissolved inorganic nitrogen was in the form of NH4-N in influent and NOX-N in effluent wastewaters, respectively (Fig. 2 and 3). Most NH4-N and NOX-N were removed by Chlorella sp., but for undissolved nitrogen was ineffective. The removal rate of NH4-N was higher than 83% in influent wastewater, especially under the 75%, 50%, 25% wastewater conditions, in which the final values were under detection limit (0.05 mg l-1) (Fig. 2). In effluent wastewater, the removal effect of NH4-N was not as good as in influent wastewater, but similar results were reported by Gonzlez et al. (1997). On the contrary, the majority of NOX-N was removed in effluent wastewater and the removal rate was more than 88% although its final content was still high (Fig. 3). Removal of phosphorus : The removal rate of total phosphorus (TP) is shown in Table 3. More than 90% TP in influent and 60% in effluent wastewaters were removed, respectively. It seems that Chlorella sp. could use phosphorus at an extremely low concentration. This phenomenon was also found in another green alga Botryococcus braunii when it was cultivated in secondarily treated sewage (Sawayama et al., 1992). A study of the growth of B. braunii in secondarily treated piggery wastewater showed its growth was nearly independent of initial phosphate concentration (An et al., 2003). A number of algae can be capable of rapidly absorbing phosphate and this surplus phosphate is usually stored as polyphosphate granules; accumulated phosphate in phosphate-rich medium can successfully sustain growth when the extracellular phosphate is very low or exhausted (Casadevall et al., 1985; Sawayama et al., 1992). Our TP
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0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 2 4 6 8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

C. Wang et al.
(a)
100% 75% 50% 25%

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

(a)
100% 75% 50% 25%

8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

0.6 0.5 0.4 0.3 0.2 0.1 0.0

(b)
100% 75% 50% 25%

8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

26 24 22 20 18 16 14 12 10 8 6 4 2 0

(b)
100% 75% 50% 25%

8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

Fig. 1 : Grow curves of Chlorella sp. in (a) influent and (b) effluent. Values are mean of three replicates + S.E.
16 14 12 10 8 6 4 2 0 0 2 4 6 8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

Fig. 3 : NOX-N in the culture medium of Chlorella sp. in (a) influent and (b) effluent. Values are mean of three replicates + S.E.
0.7

(a)
100% 75% 50% 25%

(a)
100% 75% 50% 25%

0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 2 4 6

8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

8 7 6 5 4 3 2 1 0

(b)
100% 75% 50% 25%

8 7 6 5 4 3 2 1 0

(b)

100% 75% 50% 25%

8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

8 10 1 2 14 1 6 1 8 20 2 2 24 Incubation time (days)

Fig. 2 : NH4-N in the culture medium of Chlorella sp. in (a) influent and (b) effluent. Values are mean of three replicates + S.E.

Fig. 4 : PO4-P in the culture medium of Chlorella sp. growth in (a) influent and (b) effluent. Values are mean of three replicates + S.E.

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Nutrient removal from wastewater by microalga removal rate was higher than that of Sreesai and Pakpain (2007), who reported approximately 55% of TP removal by Chlorella vulgaris in septage effluent wastewater. After 24 days of incubation, more than 90% PO4-P was removed in all treatments (Fig. 4). During the first 14 days of culture, the PO4-P was nearly exhausted in 50% influent wastewater and 50% effluent in which algae grew faster than other treatments. So, it seems that PO4-P is not the limiting factor for Chlorella sp. growth in the present culture systems. Our findings are in agreement with the results of Li et al. (2010), who found another green alga ( Scenedesmus sp.) could grow well under very low concentration of phosphorus. Therefore, the use of Chlorella sp. culture in wastewater to reduce nutrients and produce microalgae biomass is a promising approach for the production of renewable energy as an additional benefit from wastewater treatment. Acknowledgments This study was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (KZCX2-YW-QN401), the National Natural Science Foundation of China (31172114), the Natural Science Foundation for Distinguished Young Scholars of Fujian Province (2012J06009), and the International Science and Technology Cooperation Program of China (2011DFB91710). References
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