Effects of hydraulic retention time, temperature, and effluent recycling on efficiency of anaerobic filter in treating rural domestic wastewater

John Leju Celestino LADU*, Xi-wu LÜ

1. School of Energy and Environment, Southeast University, Nanjing 210096, P. R. China

2. College of Natural Resources and Environmental Studies, University of Juba, Juba 82, South Sudan

Effects of hydraulic retention time, temperature, and effluent recycling on efficiency of anaerobic filter in treating rural domestic wastewater

John Leju Celestino LADU*1,2, Xi-wu LÜ1

1. School of Energy and Environment, Southeast University, Nanjing 210096, P. R. China

2. College of Natural Resources and Environmental Studies, University of Juba, Juba 82, South Sudan

With rural population expansion and improvement of the socio-economic standard of living, treatment of rural domestic wastewater has rapidly become a major aspect of environmental concern. Selection of a suitable method for treatment of rural domestic wastewater depends on its efficiency, simplicity, and cost-effectiveness. This study investigated the effects of hydraulic retention time (HRT), temperature, and effluent recycling on the treatment efficiency of an anaerobic filter (AF) reactor. The first round of experimental operations was run for three months with HRTs of one, two, and three days, temperatures of 18℃, 21℃, and 24℃, and no effluent recycling. The second round of experimental operations was conducted for another three months with HRTs of three and four days; temperatures of 30.67℃, 30.57℃, and 26.91℃; and three effluent recycling ratios of 1:1, 1:2, and 2:1. The first round of operations showed removal rates of 32% to 44% for COD, 30% to 35% for TN, 32% to 36% for, 19% to 23% for, and 12% to 22% for TP. In the second round of operations, the removal rates varied from 75% to 81% for COD, 35% to 41% for TN, 31% to 39% for, 30% to 34% for, and 41% to 48% for TP. The average gas production rates were 6.72 L/d and 7.26 L/d for the first and second rounds of operations, respectively. The gas production rate increased in the second round of operations as a result of applied effluent recycling. The best removal efficiency was obtained for an optimum HRT of three days, a temperature of 30℃, and an effluent recycling ratio of 2:1. The results show that the removal efficiency of the AF reactor was affected by HRT, temperature, and effluent recycling.

anaerobic filter (AF); rural domestic wastewater; hydraulic retention time (HRT); effluent recycling; experimental operation

1 Introduction

Due to urbanization, changing attitudes, education, and improved socio-economic standards of living all over China, in particular in the eastern part of China, domestic wastewater is becoming a matter of concern. In the past, people lived in relatively small population units, often in the countryside, and the disposal of domestic wastewater was not devastating. However, with the expansion of rural populations, disposal and accumulation ofdomestic sewage products rapidly became one of the major aspects of environmental concern, due to the pollution of water sources, soil, and air, and, consequently, adverse impacts on the total environment. Hence, to cope with these alarming threats, an efficient, simple, cost-effective, and feasible means of anaerobic domestic wastewater treatment is highly recommended.

Many advanced anaerobic treatment reactors have been invented to tackle the challenges faced in efficient treatment of domestic wastewater. Among the reactors are the anaerobic filter (AF) reactor (Ladu et al. 2012; Chernicharo and Machado 1998; Bodik et al. 2000, 2003), the upflow anaerobic sludge blanket (UASB) reactor (Heertjes and Van Der Meer 1978; Lettinga and Vinken 1980; Lettinga et al. 1983), the hybrid reactor (Elmitwalli et al. 2002a, 2002b; Chernicharo and Nascimento 2001), the anaerobic baffled reactor (Langenhoff and Stuckey 2000; Bodik et al. 2003), the anaerobic migrating blanket reactor (Angenent and Sung 2001), and the expanded granular sludge bed reactor (Van der Last and Lettinga 1992; Seghezzo 1997).

Anaerobic processes have been used in the treatment of wastewater and sludge for more than 100 years (Martina 2008). Despite the application of these processes since the 19th century, anaerobic wastewater treatment has been considered to be inefficient and unstable. However, as a result of escalating energy prices during the 1970s and improvement in environmental education, the anaerobic treatment process has gained momentum, and more stable and efficient high-rate anaerobic systems have been developed, including AF, UASB, and the fluidized bed reactor (Gijzen 2002). The anaerobic method is now considered to be the best method for the treatment of rural domestic wastewater. Treatment of wastewater by the anaerobic process is considered sustainable (Lettinga 1996; Hammes et al. 2000) and appropriate for on-site wastewater treatment (Zeeman and Lettinga 1999) because of its simplicity and cost effectiveness, and the fact that it requires only a small space for operation.

Among the anaerobic processes, the AF process has gained popularity and has been applied in various rural domestic wastewater treatment schemes since it was first developed by Young and McCarty (1969). An AF reactor is chiefly a column or tower packed with support filter material for the growth of biomass. Its process is simple, it has a low running cost, and it does not require complex professional management. Furthermore, the process has the advantage of producing methane gas, a high-calorie fuel gas (Chernicharo 2007).

Several studies have been conducted to evaluate the performance of AF reactors, including the influence of operating characteristics and packing media (Manariotis and Grigoropoulos 2006), in treating domestic or low-strength wastewater. The effect of HRT, temperature, and effluent recycling on pollutant removal performance of the AF reactor has been examined by several researchers (Elmitwalli et al. 2002b; Bodik et al. 2000; Yu et al. 2002). Important parameters affecting the performance of the AF reactor are the operating HRT (Chuan 2007) and temperature. When the temperature is high, the conversion rates of organic matter in the anaerobic process are also high (Luostarinen et al. 2007), and at the sametime, low temperature leads to low gas production in upflow reactors and insufficient mixing. Generally, the efficiency of the anaerobic treatment process is temperature-dependent (Bogte et al. 1993; Van Haandel and Lettinga 1994). When the temperature is low, more organic matter usually remains un-degraded as a result of slow hydrolysis of volatile solids at a given HRT (Seghezzo 2004). Other studies show that, at low temperatures, longer HRTs are needed because of the lower rate of hydrolysis in AF reactors (Van der Last and Lettinga 1992). Effluent recycling has been widely used in anaerobic treatment (Sam-Soon et al. 1991). However, the effects of HRT, temperature, and effluent recycling on the removal efficiencies of anaerobic filters in treating rural domestic wastewater have been contentious. Thus, the aim of this study was to examine the effect of these factors (HRT, temperature, and effluent recycling) on the overall performance of the AF reactor in treating rural domestic wastewater at the Taihu Lake Environmental Engineering Research Center of Southeast University, Wuxi, China.

2 Materials and methods

2.1 Experimental setup

The experimental system was comprised of a regulating tank (influent tank), two submersible pumps, and an AF bioreactor system. A schematic diagram of the experimental setup is shown in Fig. 1. The AF reactor system was composed of four columns made of polyvinyl chloride (PVC), with a total capacity of 300 L. The first three columns were typically operated as an anaerobic zone and the fourth column contained an anoxic zone. The first two columns aimed mainly at the removal of physical solids in order to provide good processing conditions for the functioning of the third column. Usually, the second and third columns were optimized for volatile fatty acid (VFA) removal (Wang 1994). The AF reactors were connected in a series. The anaerobic columns had an internal diameter of 0.2 m and a height of 3 m, and the anoxic column had a diameter of 0.2 m and a height of 2 m. The columns was filled with non-woven fabric filter material with a length of 2.5 m, width of 50 mm, surface area of 150 m2/m3, and porosity of 97%. The AF reactors had an effective volume of 90 L for each anaerobic column and 60 L for the anoxic column. The influents werepumped from the same feed tank to the bottom of the columns with the help of a pump (BT100-2J). Sampling ports were located at different heights to help in extraction of samples for experimental tests and analysis.

Fig. 1 Schematic diagram of experiment

The flow rates were 300 L/d, 150 L/d, 100 L/d, and 75 L/d corresponding to HRTs of one day, two days, three days, and four days, respectively. The concentrations of suspended solids (SS), volatile suspended solids (VSS), and grey matter were 3 156 mg/L, 1 799 mg/L, and 1 357 mg/L, respectively, and the concentration ratio of VSS to SS was 0.5. The first three months of the experimental operation were conducted with HRTs of one, two, and three days, resulting in organic loading rates (OLRs) for COD of 0.19, 0.11, and 0.07 kg/(m3·d), respectively, without effluent recycling. After assessing the response of the reactor performance to those experimental factors and conditions in the first round of operations, the reactor was then operated with HRTs of three and four days, resulting in OLRs for COD of 0.08 and 0.06 kg/(m3·d) respectively, and operated with effluent recycling in the second round of operations. In this second round of experimental operations, effluent from the anoxic column was collected in the oxic column, some of it was pumped using a pump to the influent port of the reactor column, and some of it was pumped as effluent. The recycling ratio r can be defined as the ratio of the returned flow rate Qrto that of the main influent flow rate Q0, as shown in Eq. (1):

Three effluent recycling ratios, 1:1, 1:2, and 2:1, were used for the reactor. The total duration for these experimental operations was six months.

2.2 Estimation of parameters

2.2.1 Volumetric hydraulic load

The am ount of wastewater applied daily to the reactor, per unit of volume, is termed the volumetric hydraulic load (L, m3/(m3·d)) (Martina 2008):

where Q is the flow rate (m3/d), and V is the total volume of the reactor (m3). The hydraulic retention time (τ), given in days, is expressed as

which gives

2.2.2 Volumetric organic load

The volumetric organic load (VOL) is the amount of organic matter applied daily to the reactor, per unit of volume (Martina 2008):

where LOis the volumetric organic load for COD (kg/(m3·d)), and Siis the influent substrate concentration (kg/m3).

For domestic wastewater characterized by low organic matter concentrations, the VOL value to be applied usually ranges from 2.5 to 3.5 kg/(m3·d) (Chernicharo 2007).

2.2.3 Removal efficiency of reactor

The removal of COD in the AF system refers to the difference between the influent concentration (Cinf) of COD and the effluent concentration (Ceff) of COD. Thus, the removal rate (Rr) of COD is expressed by the following equation:

This equation can also be used to calculate the efficiency of the reactors in regards to other nutrients.

2.2.4 Kinetics parameters of anaerobic reactor for anaerobic digestion process

The substrate concentration surrounding the microorganisms is crucial to evaluating kinetics parameters based on Monod-type kinetic models (Govindaradjane and Sundararajan 2013). Different researchers have stressed the salience of the total OLR in assessing process performance as well as effluent substrate concentration. In view of the fact that anaerobic microorganisms, especially methanogens, are sensitive to their environment, it is more important to consider the amount of substrate per microorganism per unit period than the effluent concentration. The total OLR (Lx) takes into account both the flow rate and the concentration of the waste, and is then defined as

where Xais the active biomass concentration in the reactor.

The relationship between the substrate utilization rate (R) and Lxbecomes

where k is the maximum specific substrate utilization rate, KLis the organic loading rate at R=k 2, and q is the specific substrate utilization rate.

A mass balance on the substrate can be written around the entire system as follows:

At steady state, Eq. (9) can be combined with Eq. (8) as follows:

Similarly, a mass balance for the biomass gives the following expression:

At steady state, Eqs. (7) and (8) are substituted for the substrate removal rate in Eq. (11) to solve for Xa:

Eqs. (10) and (12) can be used to obtain the kinetic parameters k, KL, Kd, and Y.

2.3 Wastewater used for experiment

The type of wastewater used for this study was characterized by low strength. The influent was collected from a sewage manhole at the Taihu Lake Environmental Engineering Research Center of Southeast University, Wuxi, China, and then pumped into a storage tank as influent to the AF system. The AF reactors were inoculated with sludge obtained from a municipal sewage treatment plant in Wuxi. The reactors were operated continuously for 20 days as the start-up period until the performance was stable (acclimatized). The effluent samples of every reactor were collected at the sampling ports of the reactors in separate bottles every two days and stored in a refrigerator at 5 ℃ before experimental tests in the laboratory. The pH value, temperature, and gas production rate were recorded daily. The composition of the raw domestic wastewater used in this study is shown in Table 1.

Table 1 Composition of study raw domestic wastewater

2.4 Analytical procedures

The experimental test was carried for a period of six months (April, May, June, July, August, and September, 2012). All the analyses were carried out in accordance with the Chinese standard methods in Determination of Municipal Sludge in Wastewater Treatment Plant (CJ/T 221―2005). The samples were filtered througha 0.45-μm membrane filter before experimental analysis. The influent and effluent COD, TN, NH+4-N, NO3−-N, and TPconcentrations were measured according to the standard methods recommended by the U.S. Environmental Protection Agency (Clescerl et al. 1998). Temperature and pH values were measured with a dissolved oxygen meter and pH meter, respectively. The gas production rate was measured with a wet gas meter. The flow rate was controlled by a valve and incessantly regulated with the help of a pump.

3 Results and discussion

3.1 COD removal

The average concentrations of COD in the influent and effluent of the AF system were, in the first round of experimental operations, 203.07 mg/L and 123.53 mg/L, respectively, and, in the second round of experimental operations, 234.03 mg/L and 50.33 mg/L, respectively. The results provided in Tables 2 and 3 show the effects of the HRT, OLR, temperature, and effluent recycling on the removal of COD.

Table 2 Influent and effluent COD concentrations and removal efficiencies in first round of experimental operations and experimental conditions

Table 3 Influent and effluent COD concentrations and removal efficiencies in second round of experimental operations and experimental conditions

The COD removal efficiency throughout the treatment process in the first round of experimental operations was between 32% to 44% under the conditions of HRTs of three days, two days, and one day; OLRs for COD of 0.07 to 0.19 kg/(m3·d); temperatures ranging from 18 to 24℃; and without effluent recycling.

The experimental results show that HRT, temperature, OLR, and effluent recycling have a significant influence on COD removal. In the first round of experimental operations, reducing HRTs from three to two days, and then to one day increases OLR and thus affects the efficiency, resulting in COD removal rates of 44%, 40%, and 32%, respectively. That is, when the HRT decreases, the COD removal rates also decrease, and this was found to be independent of the influent concentrations.

In the second round of experimental operations, an increase in HRT and temperature andthe use of recycling led to improved efficiency of the reactor, in the range from 75% to 81% (Table 3). The maximum COD removal rate was 81%, which corresponds to an influent COD of 208.16 mg/L at a HRT of four days. The removal efficiency obtained coincided with the study conducted by Barbosa and Sant’Anna (1989), which achieved a COD removal efficiency of 74% when treating municipal wastewater at temperatures between 18℃ and 28℃, and also coincided with a study by Yu and Fang (2002), which obtained a COD removal efficiency of 89.2% without recycling and a COD removal efficiency of 92.5% with recycling. A study by Foresti (2001) showed that, at an average temperature of 25℃ and an HRT value lower than six hours, there was no significant difference in the anaerobic system performance at other temperatures and HRTs.

Comparison of the two rounds of operations shows that the COD removal efficiency of the anaerobic reactor increased with an increase in HRT and effluent recycling, and decreased with an increase in OLR, and an increase in temperature always resulted in improved removal efficiency. This agreed with the findings of Lettinga et al. (1981), who reported a COD removal rate of 65% at high temperatures in a UASB reactor, which decreased to a removal rate of 55% when the temperature was reduced to between 13℃ and 17℃.

3.2 TN removal

The results in Tables 4 and 5 illustrate the effects of the HRT, nitrogen volumetric loading rate (VLR), temperature, and effluent recycling on the removal efficiency of total nitrogen (TN) in the AF reactor. The average TN concentrations in the influent and effluent in the first round of experimental operations were 31.11 and 20.85 mg/L, respectively, and the average TN concentrations in the influent and effluent in the second round of experimental operations were 30.49 mg/L and 18.97 mg/L, respectively. These results indicate insignificant TN removal with regard to the operating conditions. This may be attributed to the low sludge production in the AF reactor, as reported by Barbosa and Sant’Anna (1989).

Table 4 Influent and effluent TN concentrations and removal efficiencies in first round of experimental operations and experimental conditions

Table 5 Influent and effluent TN concentrations and removal efficiencies in second round of experimental operations and experimental conditions

It seems that an increase in HRT enhances the nitrification. However, there was no significant difference in the removal efficiency of the AF reactor at different nitrogen VLRs and temperatures, except for the operational conditions with or without effluent recycling. It can be seen that, the higher the amount of nitrogen compounds in the influent is, the higher the removal efficiency is. Some studies have shown that the TN removal efficiency depends on the ratio of COD to TN concentrations (COD/TN ratio) and the shape of the reactor. About 80% of the TN removal efficiency was observed at a COD/TN ratio greater than 3.9 in a sequencing batch reactor (SBR) (Chul and Jay 2008). A study by Han (1996) reported a TN removal efficiency of 69.3% to 78.5% at a COD/TN ratio range of 4.5 to 5.8. In this study, a 37% to 41% of the total TN removal rate was obtained within a COD/TN ratio range of 7.2 to 7.5. The operation conditions without effluent recycling showed an average TN removal efficiency of 33%, while the operation conditions with effluent recycling revealed an average TN removal efficiency of 37.67%. Slight effects of temperature were observed.

3.3 NH4+-N removal

The results in Table 6 and Table 7 show the effect of HRT, temperature, and effluent recycling on the removal efficiency of. The average influent and effluent concentrations ofin the first round of experimental operations were 28.21 mg/L and 18.39 mg/L (Table 6). In the second round of experimental operations they were 29.33 mg/L and 18.73 mg/L, respectively (Table 7). The average removal efficiency forwith HRTs of one, two, and three days and without effluent recycling was 34.7%, while the average removal efficiency of the AF reactor with HRTs of four and three days and with effluent recycling forwas 36%. The results revealed insignificantremoval under these operating conditions. This can be attributed to inadequate sludge production in the reactor.

Table 6 Influent and effluentconcentrations and removal efficiencies in first round of experimental operations and experimental conditions

Table 6 Influent and effluentconcentrations and removal efficiencies in first round of experimental operations and experimental conditions

Month Temperature (℃) HRT (d) Cinf(mg/L) Ceff(mg/L) Effluent recycling ratio Removal efficiency (%) April 18.06 3 27.41 18.52 NR 32 May 21.49 2 28.20 18.13 NR 36 June 24.15 1 29.01 18.52 NR 36

Table 7 Influent and effluentconcentrations and removal efficiencies in second round of experimental operations and experimental conditions

Table 7 Influent and effluentconcentrations and removal efficiencies in second round of experimental operations and experimental conditions

Month Temperature (℃) HRT (d) Cinf(mg/L) Ceff(mg/L) Effluent recycling ratio Removal efficiency (%) July 30.67 4 29.59 18.29 1:1 38 August 30.57 3 29.30 17.91 1:2 39 September 26.91 3 29.10 19.98 2:1 31

3.4 NO3－-N removal

Table 8 Influent and effluentconcentrations and removal efficiencies in first round of experimental operations and experimental conditions

Table 8 Influent and effluentconcentrations and removal efficiencies in first round of experimental operations and experimental conditions

Month Temperature (℃) HRT (d) Cinf(mg/L) Ceff(mg/L) Effluent recycling ratio Removal efficiency (%) April 18.06 3 0.69 0.53 NR 23 May 21.49 2 0.58 0.47 NR 19 June 24.15 1 0.39 0.31 NR 21

Table 9 Influent and effluentconcentrations and removal efficiencies in second round of experimental operations and experimental conditions

Table 9 Influent and effluentconcentrations and removal efficiencies in second round of experimental operations and experimental conditions

Month Temperature (℃) HRT (d) Cinf(mg/L) Ceff(mg/L) July 30.67 4 5.20 3.43 August 30.57 3 3.27 2.30 September 26.91 3 3.59 2.48 Effluent recycling ratio Removal efficiency (%) 1:1 34 1:2 30 2:1 31

3.5 TP removal

The average influent and effluent total phosphorus (TP) c and 3.07 mg/L, respectively, in the first round of experimen 5.05 mg/L and 2.86 mg/L, respectively, in the second round of exp oncentrations were, 3.72 mg/L tal operations (Table 10), and erimental operations (Table 11).

Table 10 Influent and effluent TP concentrations and removal efficienc operations and experimental condition ies in first round of experimental s

Table 11 Influent and effluent TP concentrations and removal efficiencies in second round of experimental operations and experimental conditions

In regards to TP, the HRT, temperature, and operation condition with or without effluent recycling had an influence on the reactor’s removal efficiency. The efficiency improved with increasing HRT, temperature, and effluent recycling. In the first round of experimental operations, the temperature was low and there was no effluent recycling, and a 17% average TP removal rate was obtained, whereas in the second round of experimental operations, the temperature was high and effluent recycling was used, and a 44% average TP removal rate was obtained. This shows that temperature and effluent recycling significantly affects the reactor’s performance in TP removal but HRT has only a slight effect. The results also showed that TP removal rates increased with an increase in COD concentration, and this result coincided with a study by Mohammed and Abbas (2007). Giuseppe (2009) found that the higher the recycling ratio for the anaerobic treatment process was (within a limit based on kinetic considerations), the higher the removal efficiency of nutrients could be. This result coincided with a study by Elmitwalli and Ralf (2007), in which an experimental test was conducted with a UASB reactor treating grey water at HRTs between 6 and 16 hours and a temperature of 30℃, and removal efficiencies of 22% to 30% for TN and 15% to 21% for TP were obtained.

3.6 Variation of pH values

The pH value fluctuations in the influent and effluent of the AF reactor were monitored over time and the average pH values in the influent and effluent were 7.70 and 7.94, respectively, during the first round of experimental operations, and 7.70 and 7.84, respectively, during the second round of experimental operations (Tables 12 and 13).

Table 12 Influent and effluent pH values in first round of experimental operations

Table 13 Influent and effluent pH values in second round of experimental operations

Monitoring of pH value in the anaerobic reactor is crucial and can be helpful in detecting abnormalities of a system. In this study, the pH value of the treated domestic wastewater (effluent) was in the range of 7.68 to 8.04, indicating satisfactory conditions of the reactor. According to the literature (Stronach et al. 1986), pH values less than 6.8 or greater than 8.3 will cause souring of the reactor in the process of anaerobic digestion. The average pH value of the influent wastewater for the two experimental operations was 7.71, which is typical forwastewater with a mean pH value of 7.8, slightly higher than what reported by Chuan (2007). Several researchers have studied the effect of pH on the anaerobic treatment process (Yu and Fang 2002; Paulo et al. 2003; Hu et al. 2005) but there does not exist ample information about the influence of pH on AF’s efficiency in treating rural domestic waste. Generally, the pH throughout the AF effluent remained stable despite an increase in HRTs.

3.7 Gas production rate

The average gas production rate varying with time is shown in Tables 14 and 15. As the system stabilized, the average gas production rate varied between 5.82 and 8.39 L/d in the first round of experimental operations (Table 14) and in the range of 6.01 to 8.36 L/d in the second round of experimental operations (Table 15).

Table 14 Gas production rate in first round of experimental operations

Table 15 Gas production rate in second round of experimental operations

The gas production rate during the experimental operations was in the range of 5.82 to 8.39 L/d. In the first round of experimental operations, the average gas production rate was 6.72 L/d, and in the second round of experimental operations, the average gas production rate was 7.26 L/d. For the first round of operations, the highest gas production rate was 8.39 L/d in April, and the lowest was 5.82 L/d in May. The highest amount recorded was due to an increased HRT and the fact that there was no effluent recycling, and also due to the fact that April was the start-up season with active sludge and organic-rich compounds. Moreover, in May, the average gas production rate dropped significantly to 5.82 L/d. This may have been a result of a decreased HRT, where biomass has a higher propensity to be washed out from the AF system. This finding corresponds with the results of Chuan (2007). Lew et al. (2004) experienced a decline in the gas production rate when the temperature was low. A study by Agrawal et al. (1997) revealed a 78% decrease in the gas production rate when the temperature was reduced from 30℃ to 10℃. Generally, the average gas production rate increased in the second round of experimental operations as a result of applied effluent recycling, which coincides with the findings of Yu and Fang (2002).

4 Conclusions

Rural domestic sewage and its composition vary with seasons and the natural cyclical pattern of human activity. In this study, an experiment was carried out to treat rural domestic sewage using AF reactors, and the effects of HRT, temperature, and effluents recycling on the treatment efficiency of the AF reactor were investigated. The best and optimum removal efficiency was obtained at an HRT of three days, an effluent recycling ratio of 2:1, and an average temperature of 30℃. The concentrations of TN,, and TP were slightly influenced by HRT, but significantly affected by temperature and the operating conditions with or without effluent recycling. For all the experimental operations with effluent recycling, the results obtained were better than for the experimental operations without effluent recycling.

Thus, the application of the AF reactor in rural domestic wastewater can play a crucial part in the development of effective and feasible concepts for wastewater management, especially for people in rural low-income areas.

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(Edited by Yun-li YU)

This work was supported by the National Natural Science Foundation of China (Grant No. 51078074) and the Key Project of the Chinese Ministry of Education (Grant No. 308010).

*Corresponding author (e-mail: johnleju@yahoo.com)

Received Mar. 1, 2013; accepted Jan. 1, 2014