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ABSTRACT Macrophyte-cultivated wetlands have a strong potential not only to purify eutrophic water but also to produce biomass resources. Among a variety of macrophytes, we focused on the giant reed (Arundo donax), and its properties of phosphorus uptake, accumulation, and translocation were clarified in this study. Phosphorus uptake experiments using outdoor hydroponic culturing showed the seasonal variation of phosphorus uptake by the giant reed. Furthermore, two kinetic parameters describing the phosphorus uptake by the giant reed were obtained. Phosphorus accumulation experiments using radioactive phosphorus suggested that giant reeds accumulate the absorbed phosphorus in rhizomes, and it is then distributed to the leaves if needed. A phosphorus translocation experiment using radioactive phosphorus indicated that the decreasing of phosphorus in the leaves occurred in the order of location from the bottom to the top, which is relevant to the order of dying down of the plant leaves actually observed in this study. Based on these outcomes, a desirable management method for the giant reed cultivated in wetlands is proposed
Journal of Water and Environment Technology, Vol 7, No 2, 2009 Analysis of Phosphorus Behavior in the Giant Reed for Phytoremediation and the Biomass Production System Masaki SAGEHASHI*, Akira KAWAZOE**, Takao FUJII***, Hong-Ying HU****, Akiyoshi SAKODA*** *Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, 184-8588, Japan **(Former Affiliation) Institute of Industrial Science, The University of Tokyo, Tokyo, 153-8505, Japan ***Institute of Industrial Science, The University of Tokyo, Tokyo, 153-8505, Japan ****ESPC State Key Joint Lab., Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China ABSTRACT Macrophyte-cultivated wetlands have a strong potential not only to purify eutrophic water but also to produce biomass resources Among a variety of macrophytes, we focused on the giant reed (Arundo donax), and its properties of phosphorus uptake, accumulation, and translocation were clarified in this study Phosphorus uptake experiments using outdoor hydroponic culturing showed the seasonal variation of phosphorus uptake by the giant reed Furthermore, two kinetic parameters describing the phosphorus uptake by the giant reed were obtained Phosphorus accumulation experiments using radioactive phosphorus suggested that giant reeds accumulate the absorbed phosphorus in rhizomes, and it is then distributed to the leaves if needed A phosphorus translocation experiment using radioactive phosphorus indicated that the decreasing of phosphorus in the leaves occurred in the order of location from the bottom to the top, which is relevant to the order of dying down of the plant leaves actually observed in this study Based on these outcomes, a desirable management method for the giant reed cultivated in wetlands is proposed Keywords: giant reed, phosphorus, wetland INTRODUCTION Water treatment using macrophyte-cultivated wetlands has attracted wide attention, because such treatment not only purifies the environmental water but also produces biomass resources, which can potentially be used as a substitute for fossil fuel To date, many studies on wetlands for water treatments have been performed (e.g., Rousseau et al., 2008), and various plants such as the common reed (e.g., Wang et al., 2009), cattail (e.g., Gebremariam and Beutel, 2008; Yalcuk and Ugurlu, 2009), bulrush (e.g., Gebremariam and Beutel, 2008), rice (e.g., Zhou et al., 2009), and free-floating macrophytes (e.g., Nahlik and Mitsch, 2006) have been studied Among the variety of macrophytes cultivated in water treatment wetlands, we focused Received February 16th, 2009, Accepted May 27th, 2009 - 143 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 on the giant reed (Arundo donax), which is a perennial, very tall, gramineous plant with the rhizome as shown in Fig (Kawazoe et al., 2007) And gramine (N-(1H-indol-3-ylmethyl)-N,N-dimethylamine), which is widely used as an initial compound in the synthesis of a variety of substituted indoles, can be isolated from the giant reed (Semenov and Granik, 2004) Considering these characteristics as well as its phosphorus uptake potential and translocation activity of nutrients, we propose the carbon and phosphorus cycles system using the giant reed as shown in Fig 2, which can serve as a part of the sustainable material cycles system (Kawazoe et al., 2007) In this research, we clarify the phosphorus uptake, accumulation, and translocation properties of the giant reed And based on the property, we propose a management method of a water treatment system using the giant reed Fig - Appearances of the Giant Reed, Reed, and Silver Grass - 144 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 Fig - Carbon and Phosphorus Cycles System Using the Giant Reed MATERIALS AND METHODS Giant Reed Two different-aged giant reeds were used in this study One-year-old plants, which had little rhizomes, were purchased from a farmer, and several-years-old plants, which had thick rhizomes, were obtained from the botanical garden of the University of Tokyo Phosphorus Uptake by the Giant Reed The phosphorus uptake by the giant reed was measured by a series of outdoor experiments employing hydroponic culturing under various conditions Figure shows the experimental apparatus The surface area of the culture pot was about 154 cm2, and the planting density of the giant reed in this experiment was thus about 65 plant/m2 In hydroponic culturing, solid cultivation mediums such as soil are not included, to avoid the adsorption loss of phosphorus and other changes that occur in soil The experimental equipment was placed on the rooftop terrace of the Institute of Industrial Science, the University of Tokyo, which is equipped with a glass roof The culture broth was prepared as shown in Table (SHOKUBUTSU EIYOUGAKU JIKKEN HENSYU IINKAI, 1959) The changes in the phosphorus concentration in the culture broths were measured by the molybdenum blue method (Japan Sewage Works Association, 1984) every day The effects of age and weight of giant reeds on phosphorus uptake were estimated by using one-year-old plants and several-years-old plants The seasonal variation of phosphorus uptake by giant reeds was clarified by using one-year-old plants - 145 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 14 cm 1.5 m Fig - Table - 1.5 m 12 cm Apparatus for the Phosphorus Uptake Experiment Culture Broth used in the Phosphorus Uptake Experiment component (NH4)2SO4 Na2HPO4 12H2O KCl CaCl2 MgCl2 6H2O FeCl3 MnCl2 4H2O concentration 10 mg/L (depend on experiments) 10 mg/L mg/L mg/L mg/L 0.1 mg/L The inorganic phosphorus uptake by the plant root can be described by the following equation (Nielsen, 1972): Piout + R PiR Piin + R (1) where, Piout is the inorganic phosphorus concentration on the outside of the plant body, R is the transporter of the inorganic phosphorus, PiR is the phosphorus – transporter complex, and Piin is the inorganic phosphorus ion concentration inside the cell membrane Thus, when the phosphorus concentration in the culture medium is constant, the plant uptake rate, I [mg-P/(day plant)], is described as follows: I = I max C C + Km (2) - 146 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 where C is the phosphorus concentration in medium [mg-P/L], Imax is the maximum phosphorus uptake rate [mg-P/(day plant)], and Km is a constant with the same dimension as concentration [mg-P/L] From Eq (2), when the concentration in medium is changed, the amount of phosphorus uptake, N [mg-P/plant], can be described as follows: t t 0 N = ∫ Idt = ∫ I max C dt C + Km (3) ⎛ Km K m + C0 ⎞ = I max Δt ⎜1 + ⎜ C − C ln K + C ⎟ ⎟ n m n ⎠ ⎝ where t is the time [day], C0 and Cn (C0 ≠ Cn) are the concentration of phosphorus in culture medium when t=0 and t=n, respectively, and Δt is the time elapsed [day] From this equation, the parameters Imax and Km were calculated for each period based on the measured C0, C1, and the one day’s N using the non-linear least-squares method Phosphorus Accumulation and Translocation in the Giant Reed Phosphorus transport in the giant reed was directly measured by using radioactive phosphorus (32P) Experimental plants were cultured hydroponically in a room under fluorescent lighting of 15 watts The distance from the fluorescent lamp to the top of the giant reeds was about 0.2 m at the start of the experiment The temperature in the experimental room was kept at about 28 °C The rhizomes and the roots of giant reeds were soaked in Na2HPO4 aqueous solution (3.0 mg-P/L) containing a trace amount of Na2H32PO4 The amount of phosphorus accumulated was continuously measured by using a beta ray survey meter (ALOKA TGS-146) placed on the leaf The amount of phosphorus in the leaves was calculated based on the count of the beta ray, capture efficiency, device efficiency, and a specific value of the reagent The phosphorus accumulation was assumed to be uniform in all the leaves, and the total amount of the accumulation was evaluated from the total leaf area During the measurements, plants were shielded from beta rays by using acrylic boards of 1.0-cm thickness The experiment was carried out with lighting for the initial 72 hr, and afterward a 10-h light/14-h dark lighting cycle was repeated To confirm the effects of declines of temperature and day length on phosphorus transport in leaves, we performed a second, similar experiment In this experiment, the temperature and lighting cycle were changed to mimic the external temperature and the day-night variation of the season, respectively However, the light intensity was not changed RESULTS AND DISCUSSION Phosphorus Uptake by Giant Reed The effects of age and wet weight of the giant reed on phosphorus uptake are shown in - 147 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 Phosphorus uptake rate by Giant reed [mg-P/day/mday)] [mg-P/(m2 -surface] Fig The phosphorus uptake rate of several-year-old plants was lower than that of one-year-old plants in this weight range As mentioned before, the several-year-old plants have larger rhizomes, and it is possible that the phosphorus accumulated in rhizomes affects the phosphorus uptake A little dependency of phosphorus uptake on plant weight was also observed, especially in one-year-old plants However, the fluctuation range was relatively small in this weight range That is why we estimated the seasonal variation of phosphorus uptake based on plant number 90 80 70 60 50 40 30 20 年目の株 one-year-old 10 several-years-old 複数年目の株 0 Fig - 100 200 300 400 Wet weight of giant reed [g/plant] Effects of Age and Wet Weight of the Giant Reed on Phosphorus Uptake Figure shows the relationships between the phosphorus uptake and the initial phosphorus concentration in various periods The sizes of the one-year-old plants used in these experiments were comparable to the one-year-old plants used in the above mentioned experiments (Fig 4) The phosphorus uptake was larger when the initial phosphorus concentration was higher throughout the experiments High phosphorus uptake rates were observed in August and October The uptake was very sensitive to the climate conditions, most likely to the atmospheric temperature and sunshine The experimental results reflected this phenomenon The concentration range of this series of experiments was much higher than that commonly found in water environments The results show that giant reeds can be grown in areas that are very seriously polluted by phosphorus - 148 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 1500 Phosphorus uptake rate [mg-P/(day m2)] [mg-P/(m2day)] Phosphorus uptake rate [mg-P/(day m2)] [mg-P/(m2day)] 1500 1000 1000 500 6/4 - 6/11 7/4 - 7/5 8/11 - 8/12 500 10/29 - 10/30 11/3 - 11/4 12/25 - 12/26 0 10 20 30 40 50 Initial phosphorus concentration in culture medium [mg-P/L] Fig - 10 20 30 40 50 Initial phosphorus concentration in culture medium [mg-P/L] Dependency of Phosphorus Uptake Rate on Initial Concentration by the Giant Reed (Left: June to August; Right: October to December) The estimated Imax and Km based on each experiment (i.e., Fig 5) are shown in Fig Using these results, we simulated the seasonal variation of phosphorus uptake by an ideal one-dimensional giant reed cultivated waterway (Fig 7) In this simulation, the loading phosphorus concentration and water loading rate were assumed to be 2.5 mg-P/L and 0.6 m/day, respectively, based on data contained in the literature (Shioda et al., 1999) The change in phosphorus uptake rate was not ascribed to temperature change only (Fig 7) It is likely that other factors such as phosphorus inside of the plant body, growth stage of the plant, and the like also affect the phosphorus uptake by giant reeds 10 12/28 Date 11/28 -10 10/29 6/1 12/28 11/28 Date 10/29 9/29 8/30 7/31 -10 7/1 20 10 500 20 9/29 10 1000 30 8/30 1500 30 7/31 20 40 7/1 2000 40 Km [mg-P/L] 30 6/1 [mg-P/(m2day)] Imax [mg-P/day/m2] 2500 50 Temperature [ºC] 40 Temperature [ºC] 3000 Fig - Parameters Estimated from each Experiment (Left: Imax; Right: Km; ○: Air temperature at about 3:00 PM on a day after start of each observation; ■:Imax or Km) - 149 - 0.5 0.4 0.3 0.2 0.1 12/28 11/28 10/29 9/29 8/30 7/31 7/1 6/1 Phosphorus uptake rate per unit area of waterway [g-P/(m2 day)] Journal of Water and Environment Technology, Vol 7, No 2, 2009 date Date Fig - Calculated Phosphorus Uptake Rate in a One-Dimensional Giant Reed Cultivated Waterway based on Estimated Imax and Km (phosphorus concentration in loading water = 2.5 mg-P/L; water loading rate = 0.6 m/day) Phosphorus Accumulation and Translocation in the Giant Reed Figure shows the accumulation of phosphorus in leaves measured by using radioactive phosphorus (32P) The amount of phosphorus accumulation in leaves with lighting is much larger than that in leaves without lighting (gray zone in Fig 8), indicated that the phosphorus transport for leaves was affected by lighting The phosphorus accumulation in leaves at 10 days after the start of the experiment was 1.7 μg/( plant day), while in an experiment performed to clarify the amount of phosphorus uptake, the result was 134 μg/(plant day) This result suggested that giant reeds accumulated the absorbed phosphorus in the rhizomes and distributed it to the leaves if needed The results of another experiment showed that more than 60% of phosphorus in the giant reed body was located in the rhizomes (Kawazoe et al., 2007) Note that the above mentioned value of phosphorus accumulation in leaves (ca 1-2% in total uptake) can not apply for actual growing sites directly That is because we used relatively small giant reeds (see Fig 3) compared to those found at an actual growing site Relatively large (about to m in height (Kuwabara, 2008)) giant reeds are commonly found in the typical giant reed habitat The size of rhizomes and the number of stems growing from rhizomes are also different between this study and the natural environment This means that the ratio of the above-ground part of the reed to the rhizome is different, and the accumulation in leaves should be affected by the ratio - 150 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 Nonetheless, the value obtained in this study is important for the estimation of the accumulation in relatively small plants as well as for understanding the basic properties of phosphorus accumulation Accumulated phosphorus in leaves [μg-P/plant] 1.7 μg/(day･plant) day 264 240 216 192 168 144 120 96 72 48 24 0 Time elapsed [h] Fig - Accumulation of Phosphorus in Leaves Figure shows the phosphorus translocation property in each leaf when the above-ground part has died down Here, each leaf from the bottom to the top on the plant was assigned a number From this figure, we see that the phosphorus in each leaf decreased with temperature Furthermore, the decreasing of phosphorus occurred in the order from the bottom to the top, and this order was relevant to the dying down of the plant leaves actually observed in this study This result indicated that our experiments captured the translocation of phosphorus that accompanied the dying down, and suggested that the translocation of phosphorus possibly occurred in early September in the lower part of the giant reed - 151 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 Room Temperature at measurement (11:00AM) 25 20 15 Leaf no 12 1.5 10 Leaf no 0.5 Fig - Leaf no 12/13 11/28 11/13 10/29 10/14 9/29 -5 9/14 8/30 Newly accumulated phosphorus at each leaf [μg-P/one leaf] 2.5 30 Temperature at measurement Temperature at measurement (11:00AM) [ºC] Date Phosphorus Translocation Property in each Leaf when the Emerging Part has Died Down Management of the Giant Reed in Wetland Our experimental results suggested that the translocation of phosphorus from the above-ground part and the uptake of phosphorus from the roots occurred simultaneously after a particular season for several months in the giant reed The phosphorus accumulated in this period seemed to be utilized in the next year Based on these findings, a management method adequate to obtain the biomass of the giant reed and maintain the wetland potential is proposed as shown in Fig 10 In this method, the above-ground part of the giant reeds that will remain the following year are cropped before the start of the dying down period Then, after all of the above-ground part has died down, the whole plants including the roots and rhizomes are cropped except for the roots and rhizomes remaining for the next year Thereafter, the giant reeds grow with the rhizome extension, and the giant reed community in the wetland is regenerated - 152 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 Above-ground part of giant reeds that will be remained in the next year is cropped before start of the dying down period All of the above-ground part dies down Cropping with roots and rhizomes except for the remaining roots and rhizomes Growth with the rhizome extension Fig - 10 Proposed Cultivation and Cropping Method of the Giant Reed in Wetland CONCLUSIONS The seasonal variation of phosphorus uptake by giant reeds was observed by phosphorus uptake experiments using outdoor hydroponic culturing, and using the results, we obtained two kinetic parameters describing the phosphorus uptake dynamics The results of a phosphorus accumulation experiment using radioactive phosphorus suggested that giant reeds accumulated phosphorus in the rhizomes, and it was distributed to the leaves when it was required The results of a phosphorus translocation experiment using radioactive phosphorus indicated that the decreasing of phosphorus in the leaves was relevant to the dying down of the plant We proposed a desirable management method for giant reed cultivated wetland on the basis of these results ACKNOWLEDGEMENT This work was partly supported by Japan Science and Technology Agency (JST) National Natural Science Foundation of China (NSFC) Joint Program (Strategic International Cooperative Program) - 153 - Journal of Water and Environment Technology, Vol 7, No 2, 2009 REFERENCES Gebremariam S.Y., and Beutel M.W (2008) Nitrate removal and DO levels in batch wetland mesocosms: Cattail (Typha spp.) versus bulrush (Scirpus spp.), Ecological Engineering 34(1), 1-6 Japan Sewage Works Association (1984) GESUI SHIKEN HOUHOU -1984-, Japan Sewage Works Association, Tokyo, 649 p (In Japanese) Kawazoe A., Fujii T., Sagehashi M., and Sakoda A (2007) Carbon and Phosphorus Cycles System Using Giant Reed, Preprints of the 7th China-Japan Symposium on Water Environment, 107-111, 2007 Kuwabara, Y (1993) NIHON INEKA SYOKUBUTSU ZUFU, ZENKOKU NOUSON KYOUIKU KYOUKAI, Tokyo, 503 p (In Japanese) Nahlik A.M., and Mitsch W.J (2006) Tropical treatment wetlands dominated by free-floating macrophytes for water quality improvement in Costa Rica, Ecological Engineering 28(3), 246-257 Nielsen N E (1972) Transport kinetic concept of ion uptake from soil by plants II Concept and some theoretic considerations, Plant and Soil 37(3), 561-576 Rousseau D P L., Lesage E., Story A., Vanrolleghem P A., and De Pauw N (2008) Constructed wetlands for water reclamation, Desalination 218(1-3), 181-189 Semenov B.B., and Granik V.G (2004) Chemistry of N-(1H-indol-3-ylmethyl)-N,Ndimethylamine (Gramine): a review, Pharmaceutical Chemistry Journal 38(6), 287–310 Shioda T., Yamada K., Chiba N., and Sudo R (1999) The effect of porosity of packed media on nitrogen and phosphorus removal in reed bed treatment system, Journal of Japan Society on Water Environment 22(6), 505-510, 1999 (In Japanese) SHOKUBUTSU EIYOUGAKU JIKKEN HENSYU IINKAI (1959) SHOKUBUTSU EIYOUGAKU JIKKEN, ASAKURA SHOTEN, Tokyo, 600 p (In Japanese) Wang Y., Zhang J., Kong H., Inamori Y., Xu K, Inamori R., and Kondo T (2009) A simulation model of nitrogen transformation in reed constructed wetlands, Desalination 235(1-3), 93-101 Yalcuk A and Ugurlu A (2009) Comparison of horizontal and vertical constructed wetland systems for landfill leachate treatment, Bioresource Technology 100(9), 2521-2526 Zhou S., Nakashimada Y., and Hosomi M (2009) Nitrogen transformation in vertical flow systems with and without rice (Oryza sativa) studied with a high-resolution soil-water profile, Ecological Engineering 35(2), 213-220 - 154 - ... the biomass of the giant reed and maintain the wetland potential is proposed as shown in Fig 10 In this method, the above-ground part of the giant reeds that will remain the following year are... rhizomes remaining for the next year Thereafter, the giant reeds grow with the rhizome extension, and the giant reed community in the wetland is regenerated - 152 - Journal of Water and Environment... accumulation, and translocation properties of the giant reed And based on the property, we propose a management method of a water treatment system using the giant reed Fig - Appearances of the Giant Reed,