Treatment of Anaerobic Digester Effluent Using Acorus calamus: Effects on Plant Growth and Tissue Composition

The responses of Acorus calamus under greenhouse conditions for 56 days when exposed to three dilutions (25%, 50%, and undiluted) of anaerobic digester effluent from a swine farm were determined. Plant growth, morphology, pigments, and minerals in plant tissues as well as water quality were investigated. The plants grew well in all concentrations of anaerobic digester effluent with no statistically significant effects on plant growth and morphology, and without any toxicity symptoms. The NH4+ concentrations in leaves and roots and the NO3− concentrations in leaves as well as the nitrogen, phosphorus, and potassium concentrations in the plant tissues increased with increasing effluent concentration. The nutrients in the anaerobic digester effluent were removed effectively (NH4-N > 99% removal; PO4-P > 80% removal), with highest removal rates in the undiluted digester effluent. The removal of total suspended solids (>80% in 42 days) and chemical oxygen demand (37–53%) were lower. The dissolved oxygen concentration in the anaerobic digester effluent increased overtime, probably because of root oxygen release. It is concluded that Acorus calamus could be a promising species for treating high-strength wastewater with high nutrient concentrations, such as effluents from anaerobic digesters as well as other types of agricultural wastewaters.


Introduction
Discharge of contaminated water from animal farms is a main non-point source of pollution affecting organisms in aquatic ecosystems, and potentially human health [1,2]. For decades, intensive livestock production has increased greatly due to the rising demand for livestock products [3]. High volumes of livestock wastewater are consequently produced and appropriate animal waste management is required. Anaerobic digestion is a viable technology for treating several types of organic waste such as municipal, agricultural, industrial, and animal waste. Anaerobic digestion can both produce bioenergy in the form of methane and also clean the wastewater [4,5]. However, effluent from anaerobic digester treating e.g., waste from swine farms still has high concentrations of nutrients particular nitrogen (N) and phosphorus (P) [6][7][8][9]. In the anaerobic digester, the nitrogenous matter is transformed to ammonium nitrogen (NH 4 -N) by biodegradation processes [10], and the organic phosphorus or polyphosphate is transformed to orthophosphate (PO 4 -P) by biological reduction or assimilation of microorganisms, resulting in high concentrations of NH 4 -N and PO 4 -P in the anaerobic digester effluent [11]. Furthermore, the dissolved oxygen level in the anaerobic digester effluent is very   Leaf production rate (a), average leaf area (b), number of roots (c) and root length (d) (Mean ± SE) of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for a period of 56 days.

Pigment Contents
The concentrations of chlorophyll a, chlorophyll b, total chlorophylls and total carotenoids in the leaves were not significantly different among the treatments ( Figure 3). Also, the ratio of Chlorophyll a/b was not significantly different among the treatments (average 2.2 ± 0.0). , total chlorophylls (c) and total carotenoids (d) (Mean ± SE) in leaves of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for a period of 56 days.

Figure 2.
Leaf production rate (a), average leaf area (b), number of roots (c) and root length (d) (Mean ± SE) of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for a period of 56 days.

Pigment Contents
The concentrations of chlorophyll a, chlorophyll b, total chlorophylls and total carotenoids in the leaves were not significantly different among the treatments ( Figure 3). Also, the ratio of Chlorophyll a/b was not significantly different among the treatments (average 2.2 ± 0.0).

Pigment Contents
The concentrations of chlorophyll a, chlorophyll b, total chlorophylls and total carotenoids in the leaves were not significantly different among the treatments ( Figure 3). Also, the ratio of Chlorophyll a/b was not significantly different among the treatments (average 2.2 ± 0.0). , total chlorophylls (c) and total carotenoids (d) (Mean ± SE) in leaves of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for a period of 56 days. , total chlorophylls (c) and total carotenoids (d) (Mean ± SE) in leaves of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for a period of 56 days.

NH 4 + and NO 3 − in the Plant Tissue
The concentrations of anaerobic digester effluent significantly affected the concentrations of NH 4 + and NO 3 − in the leaves and that of NH 4 + in the roots (p < 0.05). The NH 4 + concentrations in leaves and roots increased with increasing concentrations of the anaerobic digester effluent (Figure 4a,b). The concentrations of NO 3 − in leaves were highest in plants grown on 100% anaerobic digester effluent ( Figure 4b). Nonetheless, the concentrations of NH 4 + in the rhizomes, and NO 3 − in the roots and the rhizomes did not significantly differ among treatments (Figure 4d-f).

NH4 + and NO3 − in the Plant Tissue
The concentrations of anaerobic digester effluent significantly affected the concentrations of NH4 + and NO3 − in the leaves and that of NH4 + in the roots (p < 0.05). The NH4 + concentrations in leaves and roots increased with increasing concentrations of the anaerobic digester effluent (Figure 4a,b). The concentrations of NO3 − in leaves were highest in plants grown on 100% anaerobic digester effluent ( Figure 4b). Nonetheless, the concentrations of NH4 + in the rhizomes, and NO3 − in the roots and the rhizomes did not significantly differ among treatments (Figure 4d-f). . NH4 + concentration in leaves (a), roots (c) and rhizomes (e) and NO3 − concentrations in leaves (b), roots (d) and rhizomes (f) (Mean ± SE) of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for a period of 56 days. Different letters indicate significant differences among treatments.

Nutrients and Minerals in the Plant Tissue
Both NH4 + and NO3 − concentrations in the leaves tended to increase with increasing concentrations of the anaerobic digester effluent, particularly the concentrations of NH4 + which were high in the roots. Compared with NH4 + and NO3 − in the rhizome, there was no significantly difference among treatments. However, NO3 − concentrations in the plant tissue was much higher than the NH4 + concentrations ( Figure 4).
The concentrations of anaerobic digester effluent significantly affected the concentrations of N, P, K and Mg in the plant tissue (p < 0.01). The total N concentrations in the roots and the rhizomes, K in the roots and P in the leaves, increased in the plants treated with high-strength anaerobic digester effluent (Figure 5b-d), whereas, Mg in the roots was reduced (Figure 5f). Moreover, the concentrations of anaerobic digester effluent significantly affected elemental stoichiometry in the

Nutrients and Minerals in the Plant Tissue
Both NH 4 + and NO 3 − concentrations in the leaves tended to increase with increasing concentrations of the anaerobic digester effluent, particularly the concentrations of NH 4 + which were high in the roots. Compared with NH 4 + and NO 3 − in the rhizome, there was no significantly difference among treatments. However, NO 3 − concentrations in the plant tissue was much higher than the NH 4 + concentrations ( Figure 4).
The concentrations of anaerobic digester effluent significantly affected the concentrations of N, P, K and Mg in the plant tissue (p < 0.01). The total N concentrations in the roots and the rhizomes, K in the roots and P in the leaves, increased in the plants treated with high-strength anaerobic digester effluent (Figure 5b-d), whereas, Mg in the roots was reduced (Figure 5f). Moreover, the concentrations of anaerobic digester effluent significantly affected elemental stoichiometry in the plants. The C:N ratios in the roots were significantly lowered (p < 0.001) when the plants grew on higher concentrations of anaerobic digester effluent (27.1 ± 1.6, 22.8 ± 0.2 and 18.2 ± 0.5 in 25%, 50% and 100% anaerobic digester effluent, respectively). However, the concentrations of anaerobic digester effluent did not affect total C, Ca, Fe and Mn in the plant tissue (Figure 5a,e,g,h). Similarly, the ratios of C:N in the leaves (average 16.5 ± 0.4) and the rhizomes (average 32.8 ± 4.9), C:P in the leaves, the roots and the rhizomes (average 71 ± 5, 128 ± 10 and 101 ± 27, respectively) and N:P in the leaves, the roots and the rhizomes (average 4.3 ± 0.3, 5.6 ± 0.3 and 2.8 ± 0.2, respectively) were not significantly different among treatments. and 100% anaerobic digester effluent, respectively). However, the concentrations of anaerobic digester effluent did not affect total C, Ca, Fe and Mn in the plant tissue (Figure 5a,e,g,h). Similarly, the ratios of C:N in the leaves (average 16.5 ± 0.4) and the rhizomes (average 32.8 ± 4.9), C:P in the leaves, the roots and the rhizomes (average 71 ± 5, 128 ± 10 and 101 ± 27, respectively) and N:P in the leaves, the roots and the rhizomes (average 4.3 ± 0.3, 5.6 ± 0.3 and 2.8 ± 0.2, respectively) were not significantly different among treatments.  , magnesium (f), iron (g) and manganese (h) (Mean ± SE) in leaves ( ), roots ( ) and rhizomes ( ) (Mean ± SE) of Acorus calamus grown on diluted anaerobic digester effluent (25% and 50%) and undiluted anaerobic digester effluent (100%) for 56 days. Different letters indicate significant differences among treatments.

Water Quality
After 56 days of treating the anaerobic digester effluent, the water quality generally improved. Dissolved oxygen dramatically decreased during the first two weeks (Figure 6a). The average pH of the anaerobic digester effluent was 6.3 ± 0.2 across the treatments (Figure 6b). Electrical Conductivity (EC) and Total Dissolved Solids (TDS) were slightly reduced (14% and 27% removal) in 50% diluted anaerobic digester effluent and 100% anaerobic digester effluent, respectively (Figure 6c,d). In addition, the TSS in the anaerobic digester effluent was reduced by 40-85% after 28 days and 82-98% after 42 days of treatments (Figure 6e). The COD gradually decreased, reaching a total removal of 37-57% (Figure 6f). For the nutrients, high NH 4 -N removal (>80% removal) was found in all treatments (Figure 7a) whereas the concentrations of NO 3 -N increased to 1.1, 1.6 and 2.7 mM in the 25%, 50%, and 100% anaerobic digester effluent, respectively ( Figure 7b). However, the concentration of NH 4 NO 3 -N was reduced by 15-55% within 28 days (Figure 7c). Similarly, PO 4 -P was removed by 64-84% at day 28 for all treatments (Figure 7d).

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester trea manure from a swine farm, despite the high nutrient concentrations (undiluted anaerob effluent). The high concentration of the anaerobic digester effluent did not affect pigme ) and 50% ( ) diluted anaerobic digester efflue 100% anaerobic digester effluent ( ) during 56 days of experiment.

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester trea manure from a swine farm, despite the high nutrient concentrations (undiluted anaerob effluent). The high concentration of the anaerobic digester effluent did not affect pigme ) and 50% ( ) diluted anaerobic digester efflue 100% anaerobic digester effluent ( ) during 56 days of experiment.

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester treat manure from a swine farm, despite the high nutrient concentrations (undiluted anaerob effluent). The high concentration of the anaerobic digester effluent did not affect pigmen ) during 56 days of experiment.

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester treating liquid manure from a swine farm, despite the high nutrient concentrations (undiluted anaerobic digester effluent). The high concentration of the anaerobic digester effluent did not affect pigment contents and minerals in the plant tissue. The nitrogen content was higher in the leaves compared with the roots and rhizomes. Similar results were found in Phragmites australis (Cav.) Trin. ex Steud. and Cyperus malaccensis var. brevifolius Boeckeler, which had higher N concentration in the leaves than the roots and stems [22]. As total C was similar among plant parts and treatments, the leaves had lower C:N ratios than the roots and rhizomes, and the C:N ratio in roots was reduced with increasing concentrations of anaerobic digester effluent. The average C:N ratio of the plants in this study was similar to the average C:N ratios of other aquatic plant species in natural habitats [22,23]. By nature, A. calamus prefers high nutrients as it is generally found in nutrients-rich habitats [19]. Likewise, the study of Votjíšková et al. [24] found that A. calamus can grow in wastewater with moderately high N (3.75 mM NH4-N and 3.75 mM NO3-N) and high P (1.5 mM PO4-P) concentrations producing high biomass without signs of NH4 + toxicity. Similarly, the highest concentrations of N and P in the anaerobic digester effluent of this study (NH4-N = 3.7 mM, NO3-N = 0.4 mM and PO4-P= 1.1 mM), which is slightly lower than in the cited previous study, did not affect plant growth. Hence, A. calamus can tolerate the high-strength anaerobic digester effluent.
Plants growing in the wastewater treatment systems play an important role to remove excess nutrients from the wastewater and part of nitrogen and phosphorus are removed by root uptake. Many studies have suggested that fast growing species could have high uptake capacity for the nutrients. Hence, most wetland species with fast growth rates have been selected and used for treating the wastewater [25][26][27]. In this study, the relative growth rate of A. calamus was 0.01-0.02 g g −1 d −1 and it was not affected by the strength of the anaerobic digester effluent. Moreover, A. calamus tolerated the high nutrient concentrations and did take up significant amounts of N and P from the

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester treating liquid manure from a swine farm, despite the high nutrient concentrations (undiluted anaerobic digester effluent). The high concentration of the anaerobic digester effluent did not affect pigment contents and minerals in the plant tissue. The nitrogen content was higher in the leaves compared with the roots and rhizomes. Similar results were found in Phragmites australis (Cav.) Trin. ex Steud. and Cyperus malaccensis var. brevifolius Boeckeler, which had higher N concentration in the leaves than the roots and stems [22]. As total C was similar among plant parts and treatments, the leaves had lower C:N ratios than the roots and rhizomes, and the C:N ratio in roots was reduced with increasing concentrations of anaerobic digester effluent. The average C:N ratio of the plants in this study was similar to the average C:N ratios of other aquatic plant species in natural habitats [22,23]. By nature, A. calamus prefers high nutrients as it is generally found in nutrients-rich habitats [19]. Likewise, the study of Votjíšková et al. [24] found that A. calamus can grow in wastewater with moderately high N (3.75 mM NH4-N and 3.75 mM NO3-N) and high P (1.5 mM PO4-P) concentrations producing high biomass without signs of NH4 + toxicity. Similarly, the highest concentrations of N and P in the anaerobic digester effluent of this study (NH4-N = 3.7 mM, NO3-N = 0.4 mM and PO4-P= 1.1 mM), which is slightly lower than in the cited previous study, did not affect plant growth. Hence, A. calamus can tolerate the high-strength anaerobic digester effluent.
Plants growing in the wastewater treatment systems play an important role to remove excess nutrients from the wastewater and part of nitrogen and phosphorus are removed by root uptake. Many studies have suggested that fast growing species could have high uptake capacity for the nutrients. Hence, most wetland species with fast growth rates have been selected and used for treating the wastewater [25][26][27]. In this study, the relative growth rate of A. calamus was 0.01-0.02 g g −1 d −1 and it was not affected by the strength of the anaerobic digester effluent. Moreover, A. calamus tolerated the high nutrient concentrations and did take up significant amounts of N and P from the

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester treating liquid manure from a swine farm, despite the high nutrient concentrations (undiluted anaerobic digester effluent). The high concentration of the anaerobic digester effluent did not affect pigment contents and minerals in the plant tissue. The nitrogen content was higher in the leaves compared with the roots and rhizomes. Similar results were found in Phragmites australis (Cav.) Trin. ex Steud. and Cyperus malaccensis var. brevifolius Boeckeler, which had higher N concentration in the leaves than the roots and stems [22]. As total C was similar among plant parts and treatments, the leaves had lower C:N ratios than the roots and rhizomes, and the C:N ratio in roots was reduced with increasing concentrations of anaerobic digester effluent. The average C:N ratio of the plants in this study was similar to the average C:N ratios of other aquatic plant species in natural habitats [22,23]. By nature, A. calamus prefers high nutrients as it is generally found in nutrients-rich habitats [19]. Likewise, the study of Votjíšková et al. [24] found that A. calamus can grow in wastewater with moderately high N (3.75 mM NH4-N and 3.75 mM NO3-N) and high P (1.5 mM PO4-P) concentrations producing high biomass without signs of NH4 + toxicity. Similarly, the highest concentrations of N and P in the anaerobic digester effluent of this study (NH4-N = 3.7 mM, NO3-N = 0.4 mM and PO4-P= 1.1 mM), which is slightly lower than in the cited previous study, did not affect plant growth. Hence, A. calamus can tolerate the high-strength anaerobic digester effluent.
Plants growing in the wastewater treatment systems play an important role to remove excess nutrients from the wastewater and part of nitrogen and phosphorus are removed by root uptake. Many studies have suggested that fast growing species could have high uptake capacity for the nutrients. Hence, most wetland species with fast growth rates have been selected and used for treating the wastewater [25][26][27]. In this study, the relative growth rate of A. calamus was 0.01-0.02 g g −1 d −1 and it was not affected by the strength of the anaerobic digester effluent. Moreover, A. calamus tolerated the high nutrient concentrations and did take up significant amounts of N and P from the

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester treating liquid manure from a swine farm, despite the high nutrient concentrations (undiluted anaerobic digester effluent). The high concentration of the anaerobic digester effluent did not affect pigment contents and minerals in the plant tissue. The nitrogen content was higher in the leaves compared with the roots and rhizomes. Similar results were found in Phragmites australis (Cav.) Trin. ex Steud. and Cyperus malaccensis var. brevifolius Boeckeler, which had higher N concentration in the leaves than the roots and stems [22]. As total C was similar among plant parts and treatments, the leaves had lower C:N ratios than the roots and rhizomes, and the C:N ratio in roots was reduced with increasing concentrations of anaerobic digester effluent. The average C:N ratio of the plants in this study was similar to the average C:N ratios of other aquatic plant species in natural habitats [22,23]. By nature, A. calamus prefers high nutrients as it is generally found in nutrients-rich habitats [19]. Likewise, the study of Votjíšková et al. [24] found that A. calamus can grow in wastewater with moderately high N (3.75 mM NH4-N and 3.75 mM NO3-N) and high P (1.5 mM PO4-P) concentrations producing high biomass without signs of NH4 + toxicity. Similarly, the highest concentrations of N and P in the anaerobic digester effluent of this study (NH4-N = 3.7 mM, NO3-N = 0.4 mM and PO4-P= 1.1 mM), which is slightly lower than in the cited previous study, did not affect plant growth. Hence, A. calamus can tolerate the high-strength anaerobic digester effluent.
Plants growing in the wastewater treatment systems play an important role to remove excess nutrients from the wastewater and part of nitrogen and phosphorus are removed by root uptake. Many studies have suggested that fast growing species could have high uptake capacity for the nutrients. Hence, most wetland species with fast growth rates have been selected and used for treating the wastewater [25][26][27]. In this study, the relative growth rate of A. calamus was 0.01-0.02 g g −1 d −1 and it was not affected by the strength of the anaerobic digester effluent. Moreover, A. calamus tolerated the high nutrient concentrations and did take up significant amounts of N and P from the ) during 56 days of experiment.

Discussion
Overall, A. calamus grew well within the effluent from the anaerobic digester treating liquid manure from a swine farm, despite the high nutrient concentrations (undiluted anaerobic digester effluent). The high concentration of the anaerobic digester effluent did not affect pigment contents and minerals in the plant tissue. The nitrogen content was higher in the leaves compared with the roots and rhizomes. Similar results were found in Phragmites australis (Cav.) Trin. ex Steud. and Cyperus malaccensis var. brevifolius Boeckeler, which had higher N concentration in the leaves than the roots and stems [22]. As total C was similar among plant parts and treatments, the leaves had lower C:N ratios than the roots and rhizomes, and the C:N ratio in roots was reduced with increasing concentrations of anaerobic digester effluent. The average C:N ratio of the plants in this study was similar to the average C:N ratios of other aquatic plant species in natural habitats [22,23]. By nature, A. calamus prefers high nutrients as it is generally found in nutrients-rich habitats [19]. Likewise, the study of Votjíšková et al. [24] found that A. calamus can grow in wastewater with moderately high N (3.75 mM NH 4 -N and 3.75 mM NO 3 -N) and high P (1.5 mM PO 4 -P) concentrations producing high biomass without signs of NH 4 + toxicity. Similarly, the highest concentrations of N and P in the anaerobic digester effluent of this study (NH 4 -N = 3.7 mM, NO 3 -N = 0.4 mM and PO 4 -P= 1.1 mM), which is slightly lower than in the cited previous study, did not affect plant growth. Hence, A. calamus can tolerate the high-strength anaerobic digester effluent. Plants growing in the wastewater treatment systems play an important role to remove excess nutrients from the wastewater and part of nitrogen and phosphorus are removed by root uptake. Many studies have suggested that fast growing species could have high uptake capacity for the nutrients. Hence, most wetland species with fast growth rates have been selected and used for treating the wastewater [25][26][27]. In this study, the relative growth rate of A. calamus was 0.01-0.02 g g −1 d −1 and it was not affected by the strength of the anaerobic digester effluent. Moreover, A. calamus tolerated the high nutrient concentrations and did take up significant amounts of N and P from the anaerobic digester effluent. The concentration of inorganic nitrogen in the plant tissue, particularly NO 3 − , increased when the plants were exposed to the high strength anaerobic digester effluents. A N mass balance calculation showed that approximately 40% of the nitrogen removed from the undiluted anaerobic digester effluent was accounted for by N assimilation by the plant, resulting in high total N accumulation in the plant tissue. Previous studies have shown that A. calamus had high removal efficiency for NH 4 -N (27-88%) and TN (41-63%) when it was used for treating swine digester effluent in horizontal subsurface flow-constructed wetland [8] and treating simulated wastewater in a vertical subsurface flow constructed wetland, respectively [15]. Compared with other studies, the N removal efficiency of A. calamus is similar to that of other emergent plants used for treating swine wastewater, such as T. latifolia, Schoenoplectus americanus (Pers.) Volkart ex Schinz & R. Keller, and Canna indica L. [28][29][30].
In the present study, it was found that a high P accumulation in the plant tissues of A. calamus was inversely correlated (p = 0.026) with the reduction of PO 4 -P in the anaerobic digester effluent. The highest removal of PO 4 -P was found in the undiluted anaerobic digester effluent. After 28 days of treatment, over 80% of PO 4 -P in the anaerobic digester effluent was removed and the concentration was lower than 0.2 mM. Similarly, a study by Fu [31] found that A. calamus removed 96.1% of TP when it grew on an artificial solution with 3 mg L −1 TP (PO 4 -P, approximately 0.1 mM TP) during 21 days and A. calamus had the highest TP removal efficiency compared to other plant species. In CWs, A. calamus reduced 64-67% TP from simulated wastewater in a vertical subsurface flow constructed wetland (VSFCW) [15] and 32-45% TP from digested effluent of swine wastewater in a HSFCW [8].
Dissolved Oxygen (DO) in the water is one of the most important factors for water quality improvement. The DO in the undiluted anaerobic digester effluent increased significantly during the beginning of the experiment. This could be the consequence of oxygen released from the roots. Acorus calamus generally grows in water-logged soils where oxygen is unavailable. Under such conditions, most plants develop air space tissue in the root cortex. Aerenchyma formation is an important response of the plants to provide interconnected air channels that enable gases to diffuse between the plant's organs. In this study, honeycomb aerenchyma developed in the root cortex of A. calamus and oxygen released from the roots, especially from lateral roots (data not shown). Moreover, the plants also had a high number of root and large roots, so root structure could play an important role in increasing DO in the anaerobic digester effluent. Hence, satisfactory removal efficiency of COD was observed, as approximately 37%, 50%, and 53% of the COD were removed from 100%, 50%, and 25% of anaerobic digester effluent, respectively, after 56 days of treatment. However, the COD removal efficiency was slightly lower than those reported from CWs planted with other plant species [8,28,32]. For the TSS removal, a large crowd of roots enhanced precipitation of suspended solids in the anaerobic digester effluent, so TSS dramatically decreased within 28 days, and more than 80% of TSS was removed after 42 days of treatment. This indicates that A. calamus is a species that performs well to remove TSS like other species used in constructed wetlands [28,29,32].

Anaerobic Digester Effluent
The wastewater used in the experiment was the effluent from an anaerobic digester used for treating liquid manure from a swine farm with 800 pigs in the Mae Hia campus, Chiang Mai University, Thailand. The characteristics of the anaerobic digester effluent are shown in Table 1.

Plant Culture and Experimental Set-Up
The plant stock of A. calamus was produced at the greenhouse of the Department of Biology, Faculty of Science, Chiang Mai University. At the beginning, five plants of similar size (approximately 30 g FW and 85 cm high) to the experimental plants were selected, their fresh weight measured and then dried in a ventilated oven at 60 • C to obtain their dry weight. Then, the fresh weight/dry weight ratio (FW/DW ratio) was calculated. For the experiment, twelve plants of similar size (approximately 30 g FW and 85 cm high) were selected. Their initial fresh weight and initial number of leaves were recorded. Then, the plants (n = 4) were placed in separate pots (25 cm in diameter and 22 cm in height) with drainage holes containing coarse sand. The plants were fertilized every 2 days with a standard growth medium modified from Smart and Barko [33] with 2 mM N (1 mM NH 4 -N:1 mM NO 3 -N) and 0.1 mM P added. After 7 days, each pot with the individual plant was placed in 15 L plastic buckets (34 cm in diameter and 36 cm in height) containing three different concentrations of the anaerobic digester effluent: 100%, 50% and 25% diluted anaerobic digester effluent, respectively ( Figure 8). The water surface was covered with a polyethylene sheet in order to prevent loss of water by evaporation during the experiment. The plants were placed in the greenhouse where the light regimes and temperature were 11 h light/13 h dark and 32 ± 5 • C:26 ± 5 • C day:night, for 56 days.

Plant Growth and Morphology
At day 56, all plants were harvested. The number of leaves, average leaf area, number of roots and root length were recorded. The leaf production rate (No d −1 ) was calculated as the increase in number of leaves throughout the experiment divided by the number of experiment days. Then, the

Plant Growth and Morphology
At day 56, all plants were harvested. The number of leaves, average leaf area, number of roots and root length were recorded. The leaf production rate (No d −1 ) was calculated as the increase in number of leaves throughout the experiment divided by the number of experiment days. Then, the plants were fractionated into leaves, roots and rhizomes. Half of the plant samples were dried in a freeze dryer (Alpha 1-4 LD plus, Martin Christ, Osterode, Germany) and the other plant samples were dried in a ventilated oven at 60 • C until constant weight. The leaf, root and rhizome biomass was recorded and the Relative Growth Rate (RGR, g g −1 d −1 ) was calculated by using the formula: RGR = (lnW 2 − lnW 1 )/(t 2 − t 1 ), where W 1 and W 2 are the initial and final total dry weight (g) and t 1 and t 2 are the initial and final time (days).

Pigments
Photosynthetic pigments (chlorophylls and carotenoids) in the leaves were analyzed in samples (8 mg) of finely-ground freeze-dried leaves. The samples were extracted in 96% ethanol (8 mL) in a dark room at room temperature for 24 h. Then the absorbance of the extracts was measured at 470 nm, 648.6 nm and 664.2 nm using a UV-VIS Spectrophotometer (UV-1800, Shimadzu Co., Kyoto, Japan). The concentrations of chlorophyll a, chlorophyll b, total chlorophylls and total carotenoids were calculated according to Lichtenthaler [34].

Nutrients and Minerals in Plant Tissue
The NH 4 + and NO 3 − in the leaves, roots and rhizomes were extracted using a hot water extraction method. Approximate 5 mg of finely-cut freeze-dried leaves, roots and rhizomes were placed in 15 mL of distilled water in screw-cap centrifuge tubes. Then, the sample tubes were incubated in a water bath at 80 • C for exactly 20 min. After that, 2.5 mL of the sample solutions were withdrawn, diluted with 2.5 distilled water and analyzed for NH 4 + concentration using a modified salicylate method (Quikchem Method No. 10-107-06-3-B; Lachat Instruments, Milwaukee, WI, USA). Two and a half milliliters of the sample solutions were withdrawn, diluted with 2.5 distilled water and analyzed for NO 3 − concentration using an ultraviolet (UV) method [35].
Total carbon (C) and nitrogen (N) concentrations in the leaves, roots and rhizomes were analyzed. Approximately 2 mg of finely-ground freeze-dried plant materials were analyzed using a CHNS analyzer (vario EL cube Elementar, Elementar Analysensysteme GmbH, Hanau, Germany). The concentrations of potassium (K), phosphorus (P), calcium (Ca), magnesium (Mg), iron (Fe), and manganese (Mn) in the leaves, roots, and rhizomes were analyzed from finely-ground freeze-dried plant materials. Approximately 200 mg of subsamples were digested with 4 mL of 65% HNO 3 and 2 mL of H 2 O 2 in a microwave sample preparation system (Multiwave 3000, Anton Paar GmbH, Austria) and the elemental concentrations were analyzed using inductively coupled plasmaspectrometry (Optima 2000 DV, PerkinElmer Instruments Inc., Shelton, CT, USA).

Water Analysis
The water samples (800 mL) from each treatment were collected at the beginning of the experiment and every 14 days after experimental set-up for a total period of 56 days. Electrical Conductivity (EC), Total Dissolved Solids (TDS), and pH were measured using a multi-parameter analyzer (CyberScan PC 300, Eutech Instruments Pte Ltd., Singapore). Dissolved Oxygen (DO) was analyzed by an azide modification method (4500-O C) [36]. Chemical Oxygen Demand (COD) was analyzed by a close reflux, titrimetric method (5220 C) [36]. Total Suspended Solids (TSS) was analyzed using a standard method 2540 D [36]. Then, the filtered water samples were analyzed for concentrations of ammonium nitrogen (NH 4 -N), nitrate nitrogen (NO 3 -N) and orthophosphate (PO 4 -P). Ammonium nitrogen concentration was analyzed using a modified salicylate method (Quikchem Method no. 10-107-06-3-B; Lachat Instruments, Milwaukee, WI, USA). Nitrate nitrogen concentration was analyzed by an UV-method [35]. Orthophosphate was analyzed by a stannous chloride method (4500-P D) [36].

Statistics
All data were statistically analyzed using SPSS Statistics version 17.0 (SPSS Inc., Chicago, IL, USA) [37]. The normality and homogeneity of variance were tested by using the Shapiro-Wilk test and Levene Statistic, respectively. If necessary, logarithmic or square root transformations were performed to ensure homogeneity of variance. Then, the data with normal distribution were tested by one-way analysis of variance (ANOVA) and the differences between treatments were identified using the Tukey HSD post hoc procedure at the 5% significance level. The data with non-normal distribution were tested by the Kruskal-Wallis test at 5% significance level.

Conclusions
In conclusion, the indifferences of growth, morphology and pigment contents of A. calamus under different concentrations of anaerobic digester effluent suggests that this species has the ability to grow and tolerate the high nutrient concentrations in the effluent from anaerobic digesters treating manure from swine farms. This plant also had elevated concentrations of NH 4 + , NO 3 − , TN, and TP in the plant tissues when the plant grew on higher concentrations of the effluent. Furthermore, A. calamus has high efficiencies for removing orthophosphate and inorganic nitrogen, as well as high efficiency for removing COD from the anaerobic digester effluent. Therefore, A. calamus could be suggested as an effective species for treating wastewater, particularly wastewater containing high N and P concentrations, such as effluents from anaerobic digesters treating wastes from animal farms as well as other types of agricultural wastewater.