ABSTRACT Double agar overlay (DAL) method was used to evaluate the inhibition of hydrophytes extracts on coliphages growth by the plaque forming unit (PFU) assay. The present research investigated allelopathic effects of extracts from five hydrophytes leaf, Polygonum hydropiper, Polygonum orientale, Phragmites communis, Arundo donax and Typha latifolia on two coliphages, T4 and f2. The results showed that different hydrophytes inhibit coliphage (T4 and f2) to various extents, and the inhibition of extracts from P. hydropiper was more effective on coliphage T4 and f2 than other hydrophytes. When the concentration of extracts was 0.5g·L-1, the inhibition rate of P. hydropiper leaf on coliphage T4, f2 was 91%, 93%, respectively. The EC50 of P. hydropiper leaf on coliphage T4 was 10mg·L-1，and the EC50 on coliphage f2 was 12.6mg·L-1. The EC50 on coliphage T4, f2 of T. latifolia leaf was 16 mg·L-1, 20 mg·L-1, respectively. The P. hydropiper showed the greatest inhibition on coliphage. The allelochemicals produced by P. hydropiper should be isolated and evaluated in the following research. More research should be done to evaluate potential use of P. hydropiper in wastewater virus controlling.
Journal of Water and Environment Technology, Vol. 7, No. 2, 2009 - 75 - Hydrophytes may play an important role in sewage disinfection in constructed wetlands Nannan ZHANG, Zhenyu WANG, Fengmin LI * * College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China ABSTRACT Double agar overlay (DAL) method was used to evaluate the inhibition of hydrophytes extracts on coliphages growth by the plaque forming unit (PFU) assay. The present research investigated allelopathic effects of extracts from five hydrophytes leaf, Polygonum hydropiper, Polygonum orientale, Phragmites communis, Arundo donax and Typha latifolia on two coliphages, T4 and f2. The results showed that different hydrophytes inhibit coliphage (T4 and f2) to various extents, and the inhibition of extracts from P. hydropiper was more effective on coliphage T4 and f2 than other hydrophytes. When the concentration of extracts was 0.5g·L -1 , the inhibition rate of P. hydropiper leaf on coliphage T4, f2 was 91%, 93%, respectively. The EC 50 of P. hydropiper leaf on coliphage T4 was 10mg·L -1 ，and the EC 50 on coliphage f2 was 12.6mg·L -1 . The EC 50 on coliphage T4, f2 of T. latifolia leaf was 16 mg·L -1 , 20 mg·L -1 , respectively. The P. hydropiper showed the greatest inhibition on coliphage. The allelochemicals produced by P. hydropiper should be isolated and evaluated in the following research. More research should be done to evaluate potential use of P. hydropiper in wastewater virus controlling. Keywords: hydrophytes, coliphage, allelopathy, inhibition. INTRODUCTION Since the early 1970s, the constructed wetlands (CWs) sewage treatment system was used for the first time in Germany. CWs had become an important sewage treatment technology after several decades of development. This kind of sewage treatment system had developed a large-scale application in Europe and the United States. CW was build with low investment, low cost, less energy-intensity and essential ecological functions in comparison to conventional sewage treatment systems (Sun et al., 2007, Chen et al., 2008). Based on the wetland's unique advantages, wetland sewage treatment technology had become an important status in China's environmental protection technology, and begun to apply far and wide in the whole nation. Constructed wetlands showed high efficiency in removing of nitrogen, phosphorus (Donga et al., 2007, Hafner et al., 2006, Lin et al.2008, Yang et al., 2008). Studies on CWs removing pathogenic micro-organisms were seldom reported. In CWs systems had proved the existence of a large number of pathogenic micro-organisms of the original sexually transmitted diseases (including gastrointestinal disease-causing bacteria, intestinal viruses and bacteriophage, etc.).If these pathogenic micro-organisms (including bacteria and virus) in the sewage could not be removed timely, they would cause a huge threat for human health and the environment (Qiu et al., 2003). In previous study, disinfection methods commonly used in sewage treatment: disinfector such as chlorine, sodium hypochlorite, chlorine dioxide (Shina et al., 2008, Xu et al., 2006) et al, ultra-violet (Chen et al., 2007, Templeton et al., 2005), Constructed Soil Filter (CSF) system (Kadam et al., 2008) and Address correspondence to College of Environmental Science and Engineering, Ocean University of China, Qingdao , Email: lfm01@ouc.edu.cn Received November 26th, 2008, Accepted February 13th, 2009 Journal of Water and Environment Technology, Vol. 7, No. 2, 2009 - 76 - sedimentation (Karima et al., 2004)and so on. The use of membrane bioreactor was tested for virus rejection, which was a possible way of removing pathogenic virus (Steven et al., 1998, Zhang et al., 2007, Zheng et al., 2007). However, because of the disadvantages: reactor restrictions, the cost relatively high and so on, they are difficult to have a good prospect in the CWs sewage purification system. Hydrophyte is an indispensable component in CWs. Researches show that the plants can produce allelochemicals and affect on the growth of other living beings. The allelochemicals of the hydrophytes inhibited the growth of algae (Donk et al., 2002, Li et al., 2004, Nakai et al., 1999, Nan et al., 2008). The moss Pleurochaete squarrosa (Basile et al., 1998), Bidens pilosa (Farah et al., 2008), Planchonia careya (McRae et al., 2008), and Polygonum cuspidatum (Shan et al., 2008) et al. showed significant antibacterial and antifungal activities. Shen J G et al. (2007) studied the anti-tobacco mosaic virus (TMV) about Ailanthus altissma and Brucea javanica, and indicated that their extracts not only inhibited the infection of TMV, but also had the inhibition for the replication of TMV. Tan Q W (2007) indicated 2-dihydroailanthone was one of the active anti-TMV ingredients in Ailanthus altissma. The whole fruit aqueous or hydroalcoholic extracts of Punica granatum L. (Punicaceae) had proved highly active against the influenza virus (Angel et al., 2008). Three Guatemalan plant extracts from Justicia reptans, Neurolaena lobata and Pouteria viridis were found to inhibit HIV replication and their hydroalcoholic extracts resulted in reversion and gene-conversion test in microorganisms, sister chromatid exchanges, micronuclei and sperm-shape abnormality assays in mice (Bedoya et al., 2008). In short, plants play an important role in antibacterial activities and controlling virus. However, the study about allelopathic effect from hydrophytes extracts controlling virus was rarely seen in sewage disinfection in CWs. Two viruses with bacterial hosts (coliphages), T4 and f2, were used in this research as surrogates for human enteroviruses to assess the hydrophytes extract’s efficiency in virus removal. It is mainly because of the following (Zheng et al., 2007): (1) The coliphage T4 which is dsRNA, 65nm× 95nm of the body and 25nm×110nm of the tail is similar to Adenovirus, Reovirus and Rotavirus, while f2 which is line ssRNA, 24-26nm of the body is similar to Poliovirus，Coxsackievirus, Echovirus, Norwalk agent and Hepatitis A virus; (2) It is non-pathogenic to humans and can be seeded with a high concentration in tracer experiments; the assay is simple and rapid. The study researched allelopathic effect from five common hydrophytes: Polygonum hydropiper, Polygonum orientale, Phragmites communis, Arundo donax and Typha latifolia on coliphage T4 and f2. In order to remove pathogenic micro-organisms in sewage and choose hydrophytes for the sewage disinfection system in CWs, the anti-coliphage activity was investigated. MATERIALS AND METHODS Materials Coliphage T4/f2 and their host counterparts E. coli B/E. coli285 were provided by associate Professor Zheng Xiang, the ecological environment research center of the Chinese Academy of Sciences. Polygonum hydropiper, Polygonum orientale, Phragmites communis, Arundo donax and Typha latifolia were harvested from Nansi Lake. Journal of Water and Environment Technology, Vol. 7, No. 2, 2009 - 77 - Experimental methods Preparation of the hydrophytes extracts Plants were washed and leaves were dried respectively for 72h at 60 ℃, and then smashed into powder, making the concentration of 100g·L -1 respectively in the dark for 48h, then these solutions were centrifuged for 15 min at 8000 r/m, and the supernatants were filtered through 0.45μm membrane. The filtrated liquids were preserved at 4 ℃. Preparation of beef extract peptone medium Peptone 10g·L -1 ，beef extract 3g·L -1 ，sodium chloride 5 g·L -1 ，pH 7.0-7.2. The top layer of soft agar and the bottom rigid layer, respectively, contained agar of 8 and 15 g·L -1. The media was autoclaved at 121℃ for 20 minutes. Measure of the plaque forming unit (PFU) The double agar overlay (DAL) method was used to evaluate the inhibition of hydrophytes extract on coliphage. 1 ml coliphage was added into a test tube, with 9 ml aseptic water being added to get different concentrations treatments. Each treatment contained 5 replications. The different concentrations (0、0.05、0.5、5、50 g·L -1 ）of hydrophytes extracts were prepared with distilled water. 1 ml coliphage and some concentration hydrophytes extract contacted for 20 minutes. Then 0.2 ml host bacteria (E. coli B/E. coli 285) and 4 ml the top layer of soft agar were transferred into each test tube, respectively. After then, the mixture in each test tube was immediately transferred into the bottom rigid layers. Coliphage T4 and f2 were counted after being incubated at 37℃ for 4~6h. A PFU was regarded as a virus with active infections. Analytical method Inhibition rate (IR) formula of hydrophytes extracts on coliphage: IR%= (1-N/N 0 ) ×100%, N: the PFU of coliphage in treatments adding extract. N 0 : the PFU of coliphage in control. The median effective concentration (EC 50 ) values of hydrophytes extracts on coliphages were calculated through straight-line interpolation (Zhou et al., 1989). Finally, EC 50 showed by dissolved organic carbon (DOC). All data in this study were tested by means of SPSS12.0, the significance between the treated groups and the controls was analyzed by one-way ANOVA with significance set at p< 0.05 and Tukey HSD was applied at p<0.05. Table 1 - the DOC concentration of extracts from five hydrophytes leaf (unit: mg•L -1 ) P. hydropiper P. orientale P. communis A. donax T. latifolia DOC 4916 2079 4602 2078 2056 RESULTS AND DISCUSSION The effect of the different hydrophytes leaf extracts on coliphages growth When coliphages were affected by the extracts, PFU would change in the short term. The figure 1 and figure 2 indicated the inhibitive effect of the P. hydropiper leaf extracts on coliphage T4 and f2. The inhibitive effect was different for different hydrophytes leaf Journal of Water and Environment Technology, Vol. 7, No. 2, 2009 - 78 - extracts. The IR on the coliphages went up gradually with the concentration of extracts increased. The IR on coliphage T4/f2 was 2.4%/1.9% when their concentration of P. hydropiper leaf extracts was 0.05 g·L -1 ; the IR was 91%/93% when their concentration was 0.5g·L -1 and the IR was nearly 100% when the concentration was 5g·L -1 . At this time there was almost no living coliphage. The IR of other hydrophytes leaf exacts on coliphages was lower than P. hydropiper extracts. As can be seen from the figure 3, we know that the IR of P. orientale extracts on coliphage T4 went up with the concentration increased. However, P. orientale extracts on coliphage f2 (figure 4) showed promotion with the concentration increased. It was obvious that the inhibitive effect of P. communis leaf extracts on coliphage T4 (figure 5) and A. donax leaf extracts on coliphage f2 (figure 8) were on the whole to promote of low-concentration and inhibit of high-concentration. The IR of P. communis extracts on coliphage f2 (figure 6) went up with the concentration increased. The IR was low before the concentration of P. communis leaf extracts was 0.5g·L -1 , but the IR had achieved 80% when the concentration was 50g·L -1 . The figure 9 and figure 10 indicated the inhibitive effect of the T. latifolia leaf extracts on coliphage T4 and f2. The IR on coliphage T4 and f2 exceeded 60% when the concentration of T. latifolia leaf extracts was 5g·L -1 . In a word, in all hydrophytes leaf extracts, the inhibitive effect of P. hydropiper leaf extracts on coliphages was the strongest. P. hydropiper displayed the better inhibitive effect on coliphage T4 and f2. So P. hydropiper extracts had the stronger allelopathic effect. -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Polygonum hydropiper leaf extracts/g·L -1 IR/% -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Polygonum hydropiper leaf extracts/g·L -1 IR/% Fig. 1 - the IR of P. hydropiper leaf extracts on coliphage T4 Fig. 2 - the IR of P. hydropiper leaf extracts on coliphage f2 -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Polygonum orientale leaf extracts/g·L -1 IR/% -200 -160 -120 -80 -40 0 0.05 0.5 5 50 the concentration of Polygonum orientale leaf extracts/g·L -1 IR/% Fig. 3 - the IR of P. orientale leaf extracts on coliphage T4 Fig. 4 - the IR of P. orientale leaf extracts on coliphage f2 Journal of Water and Environment Technology, Vol. 7, No. 2, 2009 - 79 - -150 -100 -50 0 50 100 0.05 0.5 5 50 the concentration of Phragmites communis leaf extracts/g·L -1 IR/% -40 -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Phragmites communis leaf extracts/g·L -1 IR/% Fig. 5 - the IR of P. c o m m un i s leaf extracts on coliphage T4 Fig. 6 - the IR of P. c o m m un i s leaf extracts on coliphage f2 -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Arundo donax leaf extracts/g·L -1 IR/% -80 -60 -40 -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Arundo donax leaf extracts/g·L -1 IR/% Fig. 7 - the IR of A. donax leaf extracts on coliphage T4 Fig. 8 - the IR of A. donax leaf extracts on coliphage f2 -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Typha latifolia leaf extracts/g·L -1 IR/% -20 0 20 40 60 80 100 0.05 0.5 5 50 the concentration of Typha latifolia leaf extracts/g·L -1 IR/% Fig. 9 - the IR of T. latifolia leaf extracts on coliphage T4 Fig. 10 - the IR of T. latifolia leaf extracts on coliphage f2 Comparison of EC 50 on coliphage T4 and f2 of hydrophytes extracts The EC 50 values of the five hydrophytes tested on two coliphages varied greatly. As can be seen from the table, we can see clearly that the EC 50 values of P. hydropiper leaf on coliphage T4 and f2 were 10 mg•L -1 and 12.6 mg•L -1 , respectively. The lower EC 50 values of P. hydropiper leaf showed that it inhibited the growth of coliphage T4 and f2 at lower concentration. It agrees with the results of inhibition rate experiments. Table 2 comparisons of EC50 values of hydrophytes leaf extracts on coliphage T4 and f2 (unit: mg•L -1 ) P. hydropiper P. orientale P. communis A. donax T. latifolia T4 10 398 3981 398 16 f2 12.6 promotion 316.2 251 20 Journal of Water and Environment Technology, Vol. 7, No. 2, 2009 - 80 - CONCLUSIONS A comparative study of five hydrophytes (P. hydropiper, P. orientale, P. communis, A. donax and T. latifolia) extracts on coliphage T4 and f2 showed that： 1) In all hydrophytes, the IR of P. hydropiper on coliphage T4 and f2 was the best. 2) The IR of P. hydropiper root on coliphage T4 and f2 was the strongest, and EC 50 was 10 mg·L -1 and 12.6 mg·L -1 , respectively. In conclusion, P. hydropiper may play an important role in sewage disinfection in CWs, but its living inhibiting pathogenic micro-organisms in the ecosystem need to be further studied. REFERENCES Sun G Z, Austin D. (2007). 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