Analysis of Phosphorus Behavior in the Giant Reed for Phytoremediation and the Biomass Production System

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

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

on the giant reed (Arundo donax), which is a perennial, very tall, gramineous plant with the rhizome as shown in Fig 1 (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 - 1 Appearances of the Giant Reed, Reed, and Silver Grass

Fig - 2 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 3 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 1 (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

1.5 m 1.5 m

14 cm

12 cm

Fig - 3 Apparatus for the Phosphorus Uptake Experiment

Table - 1 Culture Broth used in the Phosphorus Uptake Experiment

The inorganic phosphorus uptake by the plant root can be described by the following

equation (Nielsen, 1972):

(1)

where, Pi out 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 Pi in 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:

m

K C

C I I

+

R

where C is the phosphorus concentration in medium [mg-P/L], I max is the maximum

phosphorus uptake rate [mg-P/(day plant)], and K m 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:

+

=

n m m n

m m

t t

C K

C K C C

K t

I

t K C

C I t I N

0 0

max

0 max 0

ln 1

d d

(3)

where t is the time [day], C 0 and C n (C 0 ≠ C n) 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 I max and K m were calculated for each period based on the

measured C 0 , C 1 , 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

Fig 4 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

0 10 20 30 40 50 60 70 80 90

1年目の株 複数年目の株

Wet weight of giant reed [g/plant]

2-surface]

one-year-old several-years-old

Fig - 4 Effects of Age and Wet Weight of the Giant Reed on Phosphorus Uptake

Figure 5 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

2 day)

500

1000

1500

0 10 20 30 40 50

6/4 - 6/11 7/4 - 7/5 8/11 - 8/12

Initial phosphorus concentration

in culture medium [mg-P/L]

0

500

1000

1500

0 10 20 30 40 50

6/4 - 6/11 7/4 - 7/5 8/11 - 8/12

Initial phosphorus concentration

in culture medium [mg-P/L]

0 500 1000 1500

0 10 20 30 40 50

10/29 - 10/30 11/3 - 11/4 12/25 - 12/26

Initial phosphorus concentration

in culture medium [mg-P/L]

0 500 1000 1500

0 10 20 30 40 50

10/29 - 10/30 11/3 - 11/4 12/25 - 12/26

Initial phosphorus concentration

in culture medium [mg-P/L]

2 )]

2 )]

Fig - 5 Dependency of Phosphorus Uptake Rate on Initial Concentration by the

Giant Reed (Left: June to August; Right: October to December)

The estimated I max and K m based on each experiment (i.e., Fig 5) are shown in Fig 6 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

0

500

1000

1500

2000

2500

3000

8 -10

0 10 20 30 40

Imax

2]

Date

0 10 20 30 40 50

8 -10

0 10 20 30 40

K m

Date

Fig - 6 Parameters Estimated from each Experiment (Left: I max ; Right: K m; ○: Air temperature at about 3:00 PM on a day after start of each observation;

■:I max or K m)

2 day)

2 day)

2 day)]

2 day)

0 0.1 0.2 0.3 0.4 0.5

date

Fig - 7 Calculated Phosphorus Uptake Rate in a One-Dimensional Giant Reed

Cultivated Waterway based on Estimated I max and K m (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 8 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 2 to 4 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

Date

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

0

1

2

3

4

5

6

7

8

0

1

2

3

4

5

6

7

8

Time elapsed [h]

1.7 μg/(day･plant)

1 day

Fig - 8 Accumulation of Phosphorus in Leaves

Figure 9 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

0 0.5 1 1.5 2 2.5 3

0 5 10 15 20 25

30

Room Temperature at measurement

(11:00AM)

Leaf no 12

Leaf no 6 Leaf no 1

Date

0 0.5 1 1.5 2 2.5 3

0 5 10 15 20 25

30

Room Temperature at measurement

(11:00AM)

Leaf no 12

Leaf no 6 Leaf no 1

Date

Fig - 9 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