PHOSPHATASE AND UREASE ACTIVITIES IN CONSTRUCTED WETLAND AND THEIR RELATIONSHIP WITH PURIFICATION OF WASTEWATER

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1 PHOSPHATASE AND UREASE ACTIVITIES IN CONSTRUCTED WETLAND AND THEIR RELATIONSHIP WITH PURIFICATION OF WASTEWATER Yue Chunlei 1, 2 *, Chang Jie 3, Ge Ying 3, Jiang Hong 1, 4 and Li Hepeng 2 1 International Institute for Earth System Science, Nanjing University, Nanjing , P. R. China 2 Institute of Ecology and Environment, Zhejiang Forestry Academy, Hangzhou , P. R. China 3 College of Life Science, Zhejiang University, Hangzhou , P. R. China 4 International Ecology Research Center, Zhejiang Forestry University, Linan 31100, P. R. China ABSTRACT The distribution of phosphatase and urease activities in substrate and their relationship with purification efficiency of wastewater was studied in a vertical/reverse-vertical flow constructed wetland designed for processing domestic wastewater. With increasing the substrate depth, phosphatase and urease activities greatly decreased. Phosphatase and urease activities in the vertical flow chamber were higher than those in the reverse-vertical flow chamber. Phosphatase activity is positively correlated to the removal rates of total nitrogen, total phosphorus, chemical oxygen demands and biochemical oxygen demands; Urease activity was positively correlated to total nitrogen. The results indicated that phosphatase and urease activities could be utilized as indicators of wastewater treatment performances in the constructed wetland system. This research offered the theoretical basis for using substrate enzymatic activity as evaluation index of purification efficiency. KEYWORDS: Constructed wetland, enzymatic activity, phosphatase, urease, purification efficiency. INTRODUCTION Constructed wetland as an ecological engineering to treat wastewater has prevailed since 1970s. In the recent years, the interest in wastewater treatment by constructed wetlands has increased, because of their reliability, cost effectiveness, versatility over conventional engineering means and capability of treating more than one type of pollutants simultaneously [1]. At present, constructed wetlands have been used successfully for improving the quality of river water [2, 3], storm water [4], oilfield drainage [5], and municipal sewage [6]. Constructed wetlands purify wastewater through an integrated combination of biological, physical and chemi- cal interactions between the plants, the substrata and the inherent microbial community. During treatment of wastewaters by constructed wetlands, high molecular weight organic pollutants are degraded to low molecular weight nutrients, utilizable by microorganisms [7]. Current knowledge of soil biochemistry suggests that mineralization is achieved via the metabolism of microorganisms and the enzyme activity of the soil [8-10]. The production of substrate enzyme is due to the results of the activities of substrate microorganisms, animals, and plant roots. In substrate enzymes, phosphatase and urease play an important role in decomposition of organic matters. Phosphatase is responsible for hydrolyzing organic phosphorus compounds to inorganic phosphorus [11]; Urease is involved in the nitrogen cycle [12]. Although there have been several studies on the enzymatic activity in the substrate of constructed wetland [13, 14], little has been known about distribution of enzymatic activity and their relationships with purification of wastewater. This research was carried out in a vertical/reversevertical flow-constructed wetland designed for processing domestic wastewater. The aim is to reveal distribution characteristics of phosphatase and urease activities in substrate of constructed wetland, and to discuss the feasibility that enzymatic activity is used to estimate treatment efficiency of constructed wetland. MATERIALS AND METHODS Research site A vertical/reverse-vertical flow-constructed wetland system (Fig.1) was established in the greening belt of the residential district in Ningbo, Zhejiang Province, China, in July 2002 to treat domestic wastewater from 311 households. This constructed wetland was twin-shaped and consisted of two chambers, each having 800 m 2 surface area. 992

2 FIGURE 1 - Diagram of integrated vertical flow constructed wetland. TABLE 1 - Quality of input water in the constructed wetland. COD cr BOD 5 NH 4 -N TN TP TSS DO ph (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) Both chambers shared a drainage layer, which was filled with gravel to a depth of 10 cm and covered by a commonly used geotextile. The vertical and reverse vertical flow chambers were filled with homogenous sand to a thickness of 45 and 35 cm, respectively, where two terrestrial species, Canna indica and Lolium perenne, were planted. The constructed wetland was continuously loaded with the domestic wastewater yielded by about 1000 peoples. Quality of input water in the constructed wetland was shown in Table 1. Hydraulic retention time was 2.1 days on average. Sampling and measurements After a whole year, this system had been relatively steady. Substrate samples were collected at 1-month intervals from July to December Five sampling locations were selected at random in the each chamber. At each location, three samples were collected at three depth intervals of 5-10cm (upper layer), 20-25cm (middle layer) and 30-35cm (bottom layer) by using soil corer, respectively. Phosphatase and urease activities were measured by p-nitrophenyl soldium phosphate method and phenol soldium hypochlorite colorimetric method, respectively [15]. In the meantime, water samples were collected from the influent and effluent of the wetland. These water samples were transported in polyethylene bottles to the laboratory where the analyses were started within 24 h of collection. Parameters of water quality measured were as follows: chemical oxygen demand (COD), biochemical oxygen demand (BOD), total nitrogen (TN), total phosphorus (TP), ammonia-nitrogen (NH 4 -N), nitrate-nitrogen (NO 3 -N), total Kjeldahl nitrogen (TKN), and total suspended solids (TSS). The methods used for the measurements were those specified by the Methods for the Examination of Water and Wastewater [16]. RESULTS AND DISCUSSION Enzymatic activity Phosphatase activity In the constructed wetland, phosphatase activity was presented in Table 2. Phosphatase activity decreased rapidly with the depth of substrate increasing. There were extremely significant differences among upper, middle and bottom layer (p<0.001). In the vertical flow chamber, mean phosphatase activity in upper level was 3.3 times as much as that in middle level; in reverse-vertical chamber, mean phosphatase activity in upper level was 3.2 times as much as that in middle level. A significant difference in phosphatase activity was detected between the vertical and reverse-vertical flow chamber (p<0.05). Phosphatase activity in upper layer in the vertical flow chamber was significantly higher than in reverse-vertical flow chamber (p< 0.05). From Table 2, it can be seen that clear monthly variation of phosphatase activity could be observed. Urease activity In the constructed wetland, urease activity was presented in Table 3. Urease activity evidently decreased with the depth of substrate increasing, which was similar to phosphatase activity. The differences in urease activity were significant among the upper, middle and bottom layer (p< 0.001) In the vertical-flow chamber, mean urease activity in the upper layer was 2.4 times as much as that in middle layer; in the reverse-vertical chamber, urease activity in 993

3 TABLE 2 - Phosphatase activity (mg phenol/100g.24h) at different layers of substrate in the constructed wetland. Values are means±se. Vertical flow chamber Reverse-vertical flow chamber 5-10cm 20-25cm 30-35cm 5-10cm 20-25cm 30-35cm July 22.43± ± ± ± ± ±0.02 August 19.71± ± ± ± ± ±0.09 September 16.07± ± ± ± ± ±0.06 October 18.22± ± ± ± ± ±0.1 November 19.8± ± ± ± ± ±0.08 December 12.56± ± ± ± ± ±0.09 Average TABLE 3 - Urease activity (mg NH 3 -N /100g.24h) at different layers of substrate in the constructed wetland. Values are means±se. Vertical flow chamber Reverse-vertical flow chamber 5-10cm 20-25cm 30-35cm 5-10cm 20-25cm 30-35cm July 28.51± ± ± ± ± ±0.5 August 29.61± ± ± ± ± ±0.6 September 24.22± ± ± ± ± ±0.3 October 27.01± ± ± ± ± ±1.2 November 21.97± ± ± ± ±1 2.43±0.3 December 19.26± ± ± ± ± ±0.4 Average TABLE 4 - Phosphatase activity in the substrate of the constructed wetland and removal rate of pollutants. Phosphatase activity Removal (%) (mg phenol/ 100g.24h) TP COD cr BOD 5 TSS July August September October November December upper layer was 2.1 times as much as that in middle layer. Mean urease activity in the vertical flow chamber was slightly higher than in the reverse-vertical flow chamber (p>0.05). The production of enzymes in substrate of constructed wetland results from the activities of substrate microorganisms, animals and plant roots. In this type of constructed wetland, plant roots and microorganisms mainly distributed in the upper level of substrate [17], which perhaps resulted in main distribution of phosphatase and urease activities in the upper space close to surface of constructed wetland. Considering distribution characteristics of enzymes, microorganisms and plant roots, the upper space of substrate should be the most efficient purification space. In this constructed wetland, the contents of nitrogen, phosphorus and organic matter was lower in the reverse-vertical flow chamber because of purification of wastewater when wastewater flowed through the vertical flow chamber, which maybe resulted in relatively low enzymatic activity in the reverse-vertical flow chamber. It was reported that soil enzymatic activity was positively correlated to contents of nitrogen, phosphorus and organic matter [18]. The difference in substrate enzymatic activity between vertical and reverse-vertical flow chamber showed that vertical flow chamber maybe play a greater role in purifying wastewater. Relationship between enzymatic activities and removal of pollutants Relationship between phosphatase activity and pollutant removal Relationship between phosphatase activity and pollutant removal was shown in Table 4. Phosphatase activity was significantly positively correlated to the removal rate of TP (r=0.95, p=0.0047), indicating that in the purification process of wastewater, especially in the removal process of phosphorus, phosphatase activity played an important role. In addition, it was also significantly positively correlated to the removal rate of COD (r=0.87, p=0.0252) and BOD 5 (r=0.99, p=0.0005), implying that increase in phosphatase activity helps to improve removal rate of COD and BOD 5. However, there was not significant correlation between phosphatase activity and TSS (r=-0.39, p=0.4426). 994

4 Relationship between urease activity and pollutant removal Relationship between urease activity and removal rates of nitrogen was shown in Table 5. Uease activity was significantly positively correlated to the removal rates of TN (r=0.82, p=0.0449) and TKN (r=0.96, p=0.0028) and COD (r=0.92, p=0.0098). This result indicated that in the purification process of wastewater, urease activity could be used as an evaluation indicator of purification efficiency. But it was not significantly correlated to the removal rates of NH 4 -N (r=0.21, p=0.6778) and NO 3 -N (r=0.34, p= ), BOD 5 (r=0.71, p=0.1131) and TSS (r=0.74, p= ). Studies on the relationship between enzymatic activity in constructed wetland and purification efficiency can offer theoretical basis for evaluation of purification efficiency by using enzymatic activity. Phosphatase activity was significantly positively correlated to the removal rate of TP, COD and BOD 5 ; Urease activity was significantly positively correlated to removal rate of TN, KN and COD removal. They could be used as indicators of evaluation of purification efficiency in this kind of constructed wetland. Some researchers found that there existed a certain difference of phosphatase and urease activities in rhizosphere between plant species [19]. So in selecting plant species for constructed wetland, phosphatase and urease activities in rhizosphere can be used as a reference index. TABLE 5 - Urease activity in the substrate of the constructed wetland and removal rate of pollutants Urease activity Removal (%) (mg NH 3 -N /100g.24h) TN KN NH 4 -N NO 3 -N COD cr SS BOD 5 July Aguest September October November December CONCLUSIONS Phosphatase and urease activities greatly decreased with increasing the substrate depth in a vertical/reversevertical flow constructed wetland designed for processing domestic wastewater. Phosphatase and urease activities in the vertical flow chamber were higher than those in the reverse-vertical flow chamber. Phosphatase activity is positively correlated to the removal rates of total nitrogen, total phosphorus, chemical oxygen demands and biochemical oxygen demands; Urease activity was positively correlated to total nitrogen. phosphatase and urease activities could be utilized as indicators of wastewater treatment performances in the constructed wetland system. ACKNOWLEDGMENTS This study was financially supported by the National Project of Scientific and Technological Supporting Programs (No.2006BAD03A1405), Zhejiang Provincial Natural Science Fund (No ), The State Key Fundamental Science Fund of China (No. 2002CB111504, 2002CB410811, 2005CB and 2005CB422208), NSF-China Project (No ), and The State Data Synthesis and Analysis Funds of China (No. 2006DKA ). We wish to thank Yueping Jiang for analyzing the samples. REFERENCES [1] Yue, C.L., Chang J., Ge, Y. and Zhu, Y.M. (2004) Treatment efficiency of domestic wastewater by vertical/reverse-vertical flow constructed wetland. Fresen. Environ. Bull., 13, [2] Coveney, M.F., Stites, D.L. and Lowe, E.F. (2002) Nutrient removal from eutrophic lake water by wetland filtration. Ecol. Eng., 19, [3] Jing, S.R., Lin, Y.F. and Lee, D.Y. (2001) Nutrient removal from polluted river water by using constructed wetlands. Bioresource Technol., 76, [4] Johengen, T.H. and Larock, P.A. (1993) Quantifying nutrient removal processes within a constructed wetland designed to treat urban storm water runoff. Ecol. Eng., 2, [5] Ji, G.D., Sun, T.H., Zhou, Q.X., Sui, X., Chang, S.J. and Li, P.J. (2002) Constructed subsurface flow wetland for treating heavy oil-produced water of the Liaohe Oilfield in China. Ecol. Eng., 18, [6] Brix, H. (1994) Constructed wetlands for municipal wastewater treatment in Europe. In: Mitsch, W.J. (Ed.) Global Wetlands: Old World and New; Elsevier: Amsterdam, pp [7] Shackle, J., Freeman, C., Reynolds, B. (2000) Carbon supply and the regulation of enzyme activity in constructed wetlands. Soil Biology & Biochemistry, 32, [8] Brix, H. and Schierup, H. (1989) The use of aquatic macrophytes in water pollution control. Ambio, 18, [9] Martens, D.A., Johanson, J.B. and Frankenberger, W.T. (1992) Production and persistence of soil enzymes with repeated addition of organic residues. Soil Science, 153,

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