A comparative study of Cyperus papyrus and Miscanthidium violaceum-based constructed wetlands for wastewater treatment in a tropical climate

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1 Water Research 38 (24) A comparative study of Cyperus papyrus and Miscanthidium violaceum-based constructed wetlands for wastewater treatment in a tropical climate Joseph Kyambadde a,b, Frank Kansiime a, Lena Gumaelius b, Gunnel Dalhammar b, * a Makerere University Institute of Environment and Natural Resources, P.O. Box 7298, Kampala, Uganda b Department of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Centre, S Stockholm, Sweden Received 2 March 23; received in revised form 22 September 23; accepted 2 October 23 Abstract The treatment efficiencies of constructed wetlands containing Cyperus papyrus L. (papyrus) and Miscanthidium violaceum (K. Schum.) Robyns (synonymous with Miscanthus violaceum (K. Schum) Pilg.) were investigated in a tropical climate (Kampala, Uganda). Papyrus showed higher ammonium-nitrogen and total reactive phosphorus (TRP) removal (75.3% and 83.2%) than Miscanthidium (61.5% and 48.4%) and unplanted controls (27.9% ammoniumnitrogen). No TRP removal was detected in control effluent. Nutrients (N and P) were significantly higher (po:15) in papyrus than Miscanthidium plant tissues. Plant uptake and storage was the major factor responsible for N and P removal in treatment line 2 (papyrus) where it contributed 69.5% N and 88.8% P of the total N and P removed. It however accounted for only 15.8% N and 3.7% P of the total N and P removed by treatment line 3 (Miscanthidium violaceum). In addition, papyrus exhibited a significantly larger (p ¼ :) number of adventitious roots than Miscanthidium. Nitrifying bacteria attached to papyrus ( MPN/g DW) and Miscanthidium roots ( MPN/g DW) and the corresponding nitrification activities were consistent with this finding. Epiphytic nitrifiers appeared more important for total nitrification than those in peat or suspended in water. Papyrus root structures provided more microbial attachment sites, sufficient wastewater residence time, trapping and settlement of suspended particles, surface area for pollutant adsorption, uptake, assimilation in plant tissues and oxygen for organic and inorganic matter oxidation in the rhizosphere, accounting for its high treatment efficiency. r 23 Elsevier Ltd. All rights reserved. Keywords: Constructed wetlands; Cyperus papyrus; Miscanthidium violaceum; Nutrients; Tropical wetlands; Wastewater treatment 1. Introduction In Uganda and other East African countries, the lake fringe swamps and other wetlands are increasingly being used to polish secondary domestic wastewater effluents from adjacent cities [1] as well as surface-run-off and raw sewage from low-income communities [2]. However, *Corresponding author. Tel.: ; fax: address: gunnel@biotech.kth.se (G. Dalhammar). these wetlands are continuously being modified for agricultural activities and infrastructure development thus hampering their treatment efficiencies [3]. Few studies have compared the effects of different plant types on specific pollutant removal efficiencies [4,5]. Past studies [6,7] described nutrient transformations and faecal coliform removal processes in the two distinct macrophyte zones dominated by Cyperus papyrus (papyrus) and Miscanthidium violaceum (Miscanthidium) in Nakivubo wetland. Azza et al. [2] and Kipkemboi et al. [8] assessed the differential permeability of papyrus /$ - see front matter r 23 Elsevier Ltd. All rights reserved. doi:1.116/j.watres

2 476 ARTICLE IN PRESS J. Kyambadde et al. / Water Research 38 (24) and Miscanthidium root mats in Nakivubo swamp, suggesting papyrus as having a greater potential to extract nutrients from wastewater due to the thin and loose root mat that allows water plant interaction than Miscanthidium whose root mat is thick and compact. The present study is part of a larger investigation into management options for enhancing biological nutrient removal from wastewater in Uganda using natural and constructed wetland technology. The aims of this particular pilot study carried out between December, 21 and August, 22 were: (a) to compare the extent of nutrient (nitrogen and phosphorus) removal from wastewater by two local plant species dominant in Nakivubo wetland, (b) to investigate the factors responsible for differential nutrient removal rates between the two macrophyte species in pilot constructed wetlands and (c) to assess the technical viability of two macrophyte-based constructed wetlands with respect to wastewater treatment. The need for the study arises from the increased wastewater generation versus constant and/or degrading wastewater treatment facilities due to urbanization and financial constraints. From the presented data we will extrapolate and speculate on viability of two emergent floating macrophyte species in biological nutrient removal processes and their potential application for wastewater treatment onsite and for small sized communities. 2. Materials and methods 2.1. Site description The pilot constructed wetland system receiving secondary effluent from Bugolobi Sewage treatment works, Kampala, was set up in December 21 at Makerere University botanical garden, Kampala, Uganda. The design was a succession through a number of reverse vertical flowcontainers in which the effluent is discharged at the bottom of the container and displaces the solution from the top of the container, which then flows to the next container (Fig. 1). The CW beds were substrate-free to allowsufficient mixing and optimal contact between wastewater, microorganisms and plant root systems. A split-plot design with treatment line (unplanted control, papyrus and Miscanthidium) as the main plot factors and block (CW1 CW6) as subplot factors was used in this study. The three replicate treatment lines 1, 2 and 3 composed of six cells of circular shape which were arranged in series. The respective diameter and depth dimensions were.58 and.82 m for CW1 (anaerobic), and.58 and.43 m for CW2 CW6. The first and second cells (CW1 and CW2) for treatment lines 2 and 3 were not planted while CW3 CW6 were planted with locally available Cyperus papyrus (treatment line 2) and Miscanthidium violaceum (treatment line 3). CW2 cells were not planted to allow for oxygenation to occur at the air water interface due to air currents and wind. All cells of treatment line 1 were not planted (control). The planting densities were 38.5 and 61.5 plants/m 2 in each constructed wetland planted with papyrus and Miscanthidium, respectively. Plants were suspended in the containers by tying them to a network of wooden pegs/supports. A monitoring program of the constructed wetlands system began 2 months after planting in order to allow the vegetation and bio-film to establish. The monitoring was carried out for a period of 5 months Loading and water analyses All units were fed with wastewater effluent collected from National Water and Sewerage Corporation (NWSC) Bugolobi Sewage Treatment Works, Kampala at a rate of.64 m 3 /day giving a hydraulic retention time of 5 days. Wastewater flow rates were adjusted manually at the inlet of CW1 units using 24 mm gate valves and measured at the outlet using a measuring cylinder and stopwatch. Physico-chemical parameters (ph, dissolved oxygen (DO), electrical conductivity (EC) and temperature) were measured in situ using portable Wissenschaftlich- Technishe Werkstatten (WTW) microprocessor probes and meters. Water sampling was done at the influent and effluent as well as along the length of the system. Ammonium-nitrogen (NH 4 -N) was analyzed following direct Nesslerization method [9]. A diazotization method was used to analyze nitrite-nitrogen (NO 2 -N), while nitrate-nitrogen (NO 3 -N) and total-nitrogen (TN) were analyzed by Cadmium reduction method and TN after digestion [9]. Total reactive phosphorous (TRP) was analyzed following the ascorbic acid method [9]. All colorimetric determinations were made using a HACH DR-4 spectrophotometer Measurement of plant biomass, nutrients and root surface area An overviewof the rhizomatous habit and the rooting structure of the two plant species is described elsewhere [6,2]. Weekly recruitment of main roots and shoots on all plants was observed during the course of the study. Each plant was examined and newly emerged main roots and newly recruited shoots counted. During examination, plants were carefully removed from the treatment system in order to avoid damage. The weight of plants in each CW unit was taken using a Salter Samson spring balance (Salter Abbey, West Midlands, UK) and recorded prior to the beginning of the study and weekly for 1 weeks during the course of the study. Replicate samples of roots and shoots were collected during the 4th and 5th months of analysis and their dry weight

3 J. Kyambadde et al. / Water Research 38 (24) Wastewater reservoir CW CW2 CW3 CW4 CW5 CW6 Effluent Wastewater reservoir 12mm gate valve CW1 CW2 CW3 CW4 CW5 CW6 Effluent Fig. 1. Schematic diagram of pilot constructed wetlands used in this study. Treatment line 1 is unplanted control while lines 2 and 3 were planted with Cyperus papyrus and Miscanthidium violaceum, respectively. proportions of N and P determined after acid digestion according to Novozamsky et al. [1]. Assuming that the root is in form of a regular cylinder and area of the root ends is negligible, root surface area was calculated as: S ¼ 2pðRLN þ rlnþ; where S is the root surface area, p is a constant (3.14), R is the average radius of main roots, L is the average length of main roots, N is the average number of main roots, r is the average radius of adventitious roots, l is the average length of adventitious roots and n is the average number of adventitious roots. ð1þ 2.4. Enumeration of nitrifying bacteria Enumeration of nitrifying bacteria in the peat, water column and on root surfaces was carried out in the first part of the planted constructed wetland (CW3) using the most probable number (MPN) technique [11]. The media used was according to Schmidt and Belser [12]. The tests were carried out in 1 ml culture tubes with screwcaps; the total test volume was 5. ml. One milliliter of each 1-fold serial dilution prepared according to Kansiime and Nalubega [6] was inoculated into five replicate tubes containing 4 ml of sterile test media, mixed and incubated at room temperature in the dark for 3 6 weeks. Five culture tubes containing only

4 478 ARTICLE IN PRESS J. Kyambadde et al. / Water Research 38 (24) test media were included as negative controls. After inoculation, the used root samples were oven dried at 13 C for dry weight determination Nitrification activity Potential nitrification activity was carried out as described by Belser and Mays [13] with slight modifications. Briefly, 1 g of root samples were incubated aerobically at room temperature in 5 ml Erlenmeyer flasks containing 3 ml of test media (1 g neutralized bacteriological peptone (oxoid), 8.5 g NaCl,.95 g ammonium sulfate, and 1.6 g sodium chlorate per liter). For water and peat samples, 1 ml was added to 2 ml of test media. In all cases, sodium chlorate was added to inhibit the conversion of nitrite to nitrate [13]. Ammonia oxidation was monitored by analyzing 1 ml samples for nitrite every 45 min using Merck Spectroquant test kit [14] and a Tecan Sunrise Absorbance Microplate Reader. A graph of nitrite concentration versus time was plotted and the slope of the linear regression was calculated to obtain potential nitrification activity. Nitrite concentrations were calculated from absorbance of samples and standard nitrite (.2 mg/l) using Beer Lambert s law Statistical analyses Statistical analysis was performed with the package MINITAB Release for Windows and included analysis of variance (ANOVA), Bartlett s and Levene s tests for homogeneity of variance and normality and Tukey s multiple comparisons for differences between means. 3. Results 3.1. Physico-chemical parameters The changes in values of the measured physicochemical variables for the three treatments (unplanted control, papyrus and Miscanthidium planted CWs) are depicted in Table 1. The differences in water ph, conductivity, temperature and dissolved oxygen (DO) between treatments were highly significant (po:5 for Table 1 Mean7standard error values of physico-chemical variables determined in pilot CWs (n ¼ 12) Treatment line Variable CW1 CW2 CW3 CW4 CW5 CW6 1 ph EC (ms/cm) Temp. ( C) DO (mg/l) NH 4 -N (mg/l) NO 2 -N (mg/l) NO 3 -N (mg/l) TN (mg/l) TRP (mg/l) ph EC (ms/cm) Temp. ( C) DO (mg/l) NH 4 -N (mg/l) NO2-N (mg/l) NO 3 -N (mg/l) TN (mg/l) TRP (mg/l) ph EC (ms/cm) Temp. ( C) DO (mg/l) NH 4 -N (mg/l) NO 2 -N (mg/l) NO 3 -N (mg/l) TN (mg/l) TRP (mg/l)

5 J. Kyambadde et al. / Water Research 38 (24) all variables). Water ph was significantly lower in papyrus (p ¼ :1) than Miscanthidium (p ¼ :6) CWs compared to the controls. Similarly conductivity was lower in papyrus (p ¼ :2) than Miscanthidium (p ¼ :3) CWs, whereas DO was lower in Miscanthidium (p ¼ :3) than papyrus (p ¼ :4) CWs. Multiple comparisons detected higher water temperature in Miscanthidium (p ¼ :1) than papyrus (p ¼ :54). The longitudinal variation of the average concentrations of ammonium-nitrogen (NH 4 -N), nitrite-nitrogen (NO 2 -N), nitrate-nitrogen (NO 3 -N), total-nitrogen (TN) and total reactive phosphorus (TRP) is presented in Table 1. The differences in concentrations of NH 4 -N, NO 3 -N, TN and TRP between the three treatments were highly significant (po:4 for all variables). In contrast no significant differences (p ¼ :281) in NO 2 -N concentrations were detected between treatments. In all treatments, nitrite-nitrogen and nitrate-nitrogen were detected but only in small concentrations. Multiple comparisons showed significantly higher NH 4 -N removal in papyrus (p ¼ :2) than Miscanthidium (p ¼ :26) CWs compared to unplanted controls. On the other hand, NO 3 -N was significantly higher in unplanted controls than papyrus (p ¼ :27) and Miscanthidium (p ¼ :2) CWs. However, multiple comparisons detected significantly high TN removal in both papyrus (p ¼ :18) and Miscanthidium (p ¼ :17) CWs that was nearly equal. Similarly, the longitudinal TRP removal was significantly higher in papyrus (p ¼ :3) than Miscanthidium (p ¼ :4) CWs in comparison with the unplanted controls Recruitment of roots, shoots and weight increment Results of recruitment of main roots and shoots are shown in Fig. 2. The root recruitment rate per CW unit was 77 and 32 roots per week for papyrus and Miscanthidium, respectively. One-way ANOVA did not detect significant differences in root recruitment rates between the two plant species (p ¼ :72) despite papyrus having a two-fold higher rate. The trend of weight increment showed a similar pattern to that of root recruitment though it did not tally with shoot recruitment (Figs. 2 and 3). One-way ANOVA showed that weight increment was significantly different (p ¼ :14) between the two plant species. The average plant weight increment was.228 and.39 kg per CW unit for papyrus and Miscanthidium, respectively. No. of new main roots recruited per CW unit Period (weeks) Papyrus Miscanthidium No. of new shoots per CW unit wk wk1 wk2 wk3 wk4 wk5 wk6 wk7 wk8 wk9 wk1 Period (weeks) Papyrus Miscanthidium Fig. 2. Average weekly recruitment of roots and shoots per CW planted with Cyperus papyrus and Miscanthidium violaceum.

6 48 ARTICLE IN PRESS J. Kyambadde et al. / Water Research 38 (24) Though shoot recruitment rates were higher in papyrus than Miscanthidium CWs (5 and 2 shoots per week), respectively, one-way ANOVA did not detect significant differences between the two plant species (p ¼ :14) Estimation of root surface area of plants Results of root surface area determinations are shown in Table 2. Papyrus exhibited a significantly larger number of adventitious roots (p ¼ :) than Miscanthidium. The mean radii of the main root of both plant species did not vary significantly (p ¼ :196). In contrast, the average length of the main root was significantly greater (p ¼ :3) in Miscanthidium than in papyrus Nitrifying bacteria The distribution of nitrifying bacteria in the different layers of the constructed wetlands is depicted in Fig. 4. Weight increment (kg) per CW unit y =.225x x R 2 =.9834 y =.15x x R 2 =.914 wk wk1 wk2 wk3 wk4 wk5 wk6 wk7 wk8 wk9 wk1 Period (weeks) Papyrus Miscanthidium Fig. 3. Average weekly increment of plant weight per CW planted with Cyperus papyrus and Miscanthidium violaceum. Table 2 Parameter measurements used to estimate root surface area of Cyperus papyrus and Miscanthidium violaceum (n ¼ 3) Parameter Papyrus Miscanthidium Mean length of main root (cm) Mean length of adventitious roots (cm) Mean number of adventitious roots/main root Mean radius of main root (cm) Mean radius of adventitious roots (cm) Average root surface area (cm 2 ) a a Calculated using Eq. (1). Log number (MPN/1 ml or gdw) 1.E+8 1.E +7 1.E+6 1.E+5 1.E+4 1.E+3 1.E+2 1.E+1 1.E+ Water Peat Papyrus roots Miscanthidium roots Sample Fig. 4. Average spatial distribution of nitrifying bacteria in water, peat and on roots of Cyperus papyrus and Miscanthidium violaceum in a pilot constructed wetland. Bars indicate standard error of the mean (n ¼ 3).

7 J. Kyambadde et al. / Water Research 38 (24) Numbers of ammonia oxidizing bacteria followed the pattern peat>papyrus>miscanthidium>water column. The mean numbers of nitrifying bacteria in the papyrus root mat ( MPN/g DW) were significantly higher (p ¼ :) than those associated with the Miscanthidium root mat ( MPN/g DW) Potential nitrification activity Results of potential microbial nitrification activities for water, peat and root mat samples of papyrus and Miscanthidium are depicted in Figs. 5a and b. Higher nitrification potential was found to be associated with papyrus roots than Miscanthidium, water or peat samples (Fig. 5a). The root mat samples of the two plant species showed similar nitrification capacities for the first 3 h. However, papyrus surpassed to attain a maximum nitrite production of.8 mg/l in the next 1.5 h. In contrast, Miscanthidium root samples attained a maximum of.514 mg/l in the first 3 h before it leveled off (Fig. 5b). Similarly, high activity was detected in the peat although it was not significantly different from that of water (p ¼ :53). The activity for peat samples reached a maximum of.213 mg nitrate-n/l in 7 h as opposed to a maximum of.183 mg nitrate-n/l in the water column samples after 5.5 h (Fig. 5b). The slopes of linear regression for water, peat and roots of papyrus and Miscanthidium were.31,.314,.147 and.146 mg NO 2 /l/h Nitrogen and phosphorus content in plant tissues Results of nutrient content in plant tissues are depicted in Fig. 6. Concentrations of N were significantly higher than P in both papyrus (p ¼ :37) and Miscanthidium (p ¼ :4) tissues. In addition, N levels were significantly higher (p ¼ :14) in papyrus than in Miscanthidium. Similarly, P levels were significantly higher (p ¼ :4) in papyrus than in Miscanthidium. N and P content (g/kg dry weight) Papyrus root Miscanthidium root Papyrus shoot Plant tissue Miscanthidium shoot Nitrogen Phosphorus Fig. 6. Nitrogen and phosphorus content in root and shoot tissues of Cyperus papyrus and Miscanthidium violaceum during the 4th and 5th months of analysis. Bars indicate standard error of the mean (n ¼ 3). Nitrite-N concentration (mg/l) Nitrite-N concentration (mg/l) y =.39x R 2 =.9886 y =.299x R 2 = Time (h) Time (h) Nitrite-water Nitrite-Peat Nitrite-water Nitrite-Peat Nitrite-N concentration (mg/l) (a) Time (min) Nitrite-N concentration (mg/l) (b) y =.27x R 2 =.9649 y =.25x R 2 = Time (min) Papyrus Miscanthidium Papyrus Miscanthidium Fig. 5. (a) Potential nitrification activities of water, peat, papyrus and Miscanthidium root samples. (b) Linear part of nitrification rates and the corresponding root square values.

8 482 ARTICLE IN PRESS J. Kyambadde et al. / Water Research 38 (24) Discussion Our qualitative observations of several quantified water quality parameters suggest a high potential of both plant species in biological nutrient removal processes. In both planted CWs, water ph was within the allowable range of for the survival of most bacteria [15]. The water ph value was slightly acidic (ph ) in CWs planted with papyrus compared to Miscanthidium (ph ; Table 1) probably due to decomposition products of wastewater components [16] trapped in the papyrus root mat as well as decomposing plant materials. Our investigation indicated a two-fold higher root recruitment rate for papyrus with many adventitious root structures than Miscanthidium (Fig. 2; Table 2), which effectively trapped organic suspended particles in feed wastewater and dead plant material. Contrary to field observations, the water ph value in Miscanthidium CWs was higher than ph 5. values reported for Miscanthidium root mats in Nakivubo wetland [17] and other wetlands on the northern shores of Lake Victoria [8]. The significant difference in water temperature between the three treatments was probably due to the small scale of the CWs. Unplanted CWs showed higher DO concentrations than the corresponding planted wetland units and this was attributed to algal photosynthesis. Effective nitrification has been reported in systems with DO content of only.5 mg/l [18]. In this study, the concentrations of nitrite and nitrate in the unplanted control systems were low despite DO concentrations higher than 2.5 mg/l. This is attributed to denitrification and lack of sufficient attachment sites and subsequent flowthrough of nitrificants. In contrast, concentrations of DO decreased in all vegetated CWs due to aerobic decomposition of plant materials, nitrification and minimal surface aeration resulting from vegetation coverage. The lownitrate levels in planted CWs can be explained by heterotrophic competition with nitrificants for oxygen [19], denitrification and plant uptake. The values of electrical conductivity and the concentrations of NH 4 -N, NO 2 -N, NO 3 -N, TN and TRP were generally reduced in all CWs. Reduction in these quantified water quality parameters was well demonstrated in planted CWs and was generally higher in papyrus than Miscanthidium CWs. TRP effluent concentrations belowthe Uganda regulatory discharge limit of 1 mg/l [2] were obtained in both planted CWs, with papyrus showing much lower concentrations of up to 2.6 mg/l. However, the effluent NH 4 -N concentration belowthe national discharge limit of 1 mg/l could only be obtained in papyrus CWs. The longitudinal NH 4 -N and TRP removal rates of 75.3% and 83.2% in papyrus and 61.5% and 48.4% for Miscanthidium-based CWs were achieved compared to only 27.9% NH 4 -N removal and 1.3% TRP accumulation in unplanted controls. Thus, both macrophytes demonstrated a positive influence on nutrient removal processes. The removal values for ammonium (75.3%) and phosphorus (83.2%) obtained in the papyrus systems were high compared to values reported by Lizhibowa [21] (7% NH 4 -N) in bucket experiments at Kiriinya [22] (o2% for both NH 4 -N and TRP) in CW pilot units at Kiriinya. Ammonium-nitrogen was removed through plant uptake and microbial nitrification/denitrification processes since temperature and ph were within the range that could support both nitrification and denitrification processes [18]. Ammonia volatilisation could not have occurred because the ph in the system never rose above ph 9 [23]. In aquatic systems, phosphorus removal is mainly via biological uptake by bacteria, phytoplankton and plants [24,25] and adsorption and precipitation by a substratum surface containing free Ca 2+, Fe 2+, Mg 2+ and Al 3+ ions [26,27]. Even though epiphytic uptake was not quantified, it could not account for the differences observed in the treatment systems since they were all exposed to similar conditions. As our design was substrate-free, immobilization and soil sorption could not influence the removal processes. However, the possibility of phosphorus precipitation, adsorption of soluble phosphorus to the roots systems and suspended solids settling at the bottom of treatment CWs could not be ruled out. Mass balance calculations showed plant uptake and storage contribution of 69.5% N and 88.8% P of the total N and P removed by papyrus treatment line and 15.8% N and 3.7% P of the total N and P removed by Miscanthidium violaceum treatment line. Therefore active uptake and incorporation into plant tissue was the major factor responsible for the observed phosphate removal rates in CWs planted with Cyperus papyrus. Other processes such as nitrification denitrification and adsorption of soluble phosphorus to roots and peat were more important for N and P removal in CWs planted with Miscanthidium violaceum. The differences in the structure and recruitment of roots by the two macrophytes depicted important consequences for the degradation of wastewater components and uptake of nutrients. Indeed, this was supported by the difference in root surface area (Papyrus, 28.6 cm 2 per main root compared to Miscanthidium, 72.2 cm 2 ) and recruitment rates between the two macrophytes. Papyrus exhibited a two-fold higher root recruitment rate than Miscanthidium. Similarly, the high number of adventitious root structures conferred papyrus a three-fold larger root surface area than Miscanthidium. Wetland plants are reported to transfer photosynthetic oxygen to the rhizosphere thus boosting oxygen concentration in the water column [28,29]. Assuming the oxygen transfer rate of papyrus to be half the maximum transfer rate (12 g/m 2 /day) reported for Phragmites australis [3], papyrus CWs provided

9 J. Kyambadde et al. / Water Research 38 (24) g O 2 /m 2 /day compared to the oxygen demand of 5.7 g/day. It is probable that the high root numbers of papyrus coupled with its larger root surface area due to adventitious root structures provided more oxygen to the rhizosphere of root mat, reducing competition between heterotrophs and nitrificants in papyrus CWs. Differences in root structures and numbers influenced periphyton attachment and material transformations in wetland systems [31,32]. In addition to providing attachment sites and diffusible oxygen to the bacteria, root mats increase wastewater residence time and retention of suspended organic particles, which upon degradation avail nutrients to bacteria and plants. In fact, more nitrifying bacteria were associated with papyrus roots ( MPN/g DW) compared to Miscanthidium roots ( MPN/g DW) and water column ( MPN/1 ml), suggesting more attachment sites and easy accessibility of nutrients. The numbers of nitrifying organisms on papyrus roots and in water column were within the range found in Nakivubo wetland [6]. The numbers of nitrifying bacteria enumerated in the peat ( MPN/1 ml) and epiphyton were comparably higher than those reported by K.orner [33]. Nitrifying bacteria in the peat might have been inactive in the treatment system and may not have contributed much to the nitrification process but only became active when conditions were favorable in our laboratory. Indeed, this was consistent with the results of potential nitrification activities of plant roots, water and peat samples. Higher activity was associated with plant roots particularly papyrus roots (Fig. 5a) and tallied with MPN numbers (Fig. 4) except for the peat samples. The larger surface area of papyrus root structures attracted more nitrifying bacteria by providing attachment sites, diffusible oxygen from the aerial parts, and substrates from trapped decomposing wastewater components. Nitrifiers in the epiphyton have been estimated to have an equally high influence on total system nitrification as those in the sediment although the peat is more important for denitrification [33]. Results from this study showed a 5-fold higher nitrification activity in the epiphyton than in the water column and sediment suggesting epiphytic nitrifiers to be more important for total system nitrification. One possible explanation is that the nitrifying bacteria in the peat were not so active due to competition for the limited oxygen with heterotrophs. However, their re-growth and activity was supported in our culture tubes where conditions were ambient. Thus, suspended nitrifying organisms in the constructed wetlands positively influenced total system nitrification bearing in mind the long retention time [34] and fairly high numbers ( MPN/ 1 ml) in the water column. Microbial attachment and root development seemed to have influenced positively differential nutrient uptake by the two macrophytes. More shoots were developed by papyrus with a corresponding increase in plant fresh weight, a possible indicator that it assimilated more nutrients than Miscanthidium. In fact, analysis of phosphorus and nitrogen content in roots and shoots of both macrophytes (Fig. 6) indicated higher concentrations of the two nutrient variables in papyrus than Miscanthidium plant tissues. 5. Conclusion To evaluate the potential application of a macrophyte in wastewater treatment constructed wetlands, knowledge of structural development and recruitment rates of roots and the general growth rate of the macrophyte in question is crucial. This influences plant microorganisms wastewater interactions by providing microbial attachment sites, sufficient wastewater residence time, trapping and settlement of suspended wastewater components as a result of resistance to hydraulic flow, surface area for pollutant adsorption, uptake and storage in plant tissues, and diffusion of oxygen from aerial parts to the rhizosphere. In this study, we observed high wastewater treatment efficiencies associated with planted CWs compared to unplanted controls. Papyrus CWs showed markedly higher nutrient removal efficiencies, with higher N and P levels in plant tissues in comparison to Miscanthidium. Similarly, more shoots were developed by papyrus than Miscanthidium possibly indicating differences in nutrient uptake. This study showed that epiphytic nitrifiers were more important for total nitrification than those in peat or suspended in water and also demonstrated that root development and its association with microorganisms is responsible for the high wastewater treatment potential of papyrus that was reported in past studies. At present, we can speculate on the potential application of papyrus for wastewater treatment especially for small-sized communities and onsite treatment and attribute the observed differential nutrient removal rates to a combination of factors stated above, but these remain the topics of our ongoing research activities. Acknowledgements This study received financial support from the Swedish International Development Cooperation Agency (Sida)/Department of Research Cooperation (SAREC) under the wastewater treatment component of the East African Regional Programme and Research Network for Biotechnology, Biosafety and Biotechnology Policy Development (BIO-EARN). Authors are

10 484 ARTICLE IN PRESS J. Kyambadde et al. / Water Research 38 (24) grateful to the Director, MUIENR for material support and Mr. Robert Bikala and Mr. Bright Twesigye for their technical support during system set-up and monitoring. References [1] Chale FMM. Plant biomass and nutrient levels of a tropical macrophyte (Cyperus papyrus L.) receiving domestic wastewater. Hydrobiol Bull 1987;21(2): [2] Azza NGT, Kansiime F, Nalubega M, Denny P. Differential permeability of papyrus and Miscanthidium root mats in Nakivubo swamp, Uganda. Aquat Bot 2;67: [3] Emerton L, Iyango L, Luwum P, Malinga A. The present economic value of Nakivubo urban wetland, Uganda. IUCN The World Conservation Union, Eastern Africa Regional Office, Nairobi and National Wetlands Programme, Wetlands Inspectorate Division, Ministry of Water, Land and Environment, Kampala, p [4] Gersberg RM, Elkins BV, Lyon SR, Golman CR. Role of aquatic plants in wastewater treatment by artificial wetlands. Water Res 1986;2(3): [5] Reddy KR, Patrick WH, Lindau CW. Nitrification denitrification at the plant root sediment interface in wetlands. Limnol Oceanogr 1989;34(6): [6] Kansiime F, Nalubega M. Wastewater treatment by a natural tropical wetland: the Nakivubo swamp, Uganda. Processes and implications. Ph.D. thesis, IHE, Delft/ Agricultural University, Wageningen, The Netherlands. Rotterdam, The Netherlands: A.A. Balkema Publishers; pp. [7] Kansiime F, van Bruggen JJA. Distribution and retention of faecal coliforms in the Nakivubo wetland in Kampala, Uganda. Water Sci Technol 21;44(11/12): [8] Kipkemboi J, Kansiime F, Denny P. The response of Cyperus papyrus (L.) and Miscanthidium violaceum (K. Schum) Robyns to eutrophication in natural wetlands of Lake Victoria, Uganda. Afr J Aquat Sci 22;27(1): [9] American Public Health Association (APHA). Standard methods for the examination of water and waste water, 18th ed. Washington, DC, USA: APHA AWWWA WEF; [1] Novozamsky I, Houba VJG, van Eck R, van Vark W. A novel digestion technique for multi-element plant analysis. Commun Soil Sci Plant Anal 1983;14(3): [11] Mutulewich VA, Strom PE, Finstein MS. Length of incubation for enumerating nitrifying bacteria present in various environments. Appl Microbiol 1975;29: [12] Schmidt EL, Belser LW. Nitrifying bacteria. In: Page AL, Miller RH, Keeney DR, editors. Methods of soil analysis Part 2: chemical and microbiological properties (2nd). Madison, WI: American Society of Agronomy Monograph, vol. 9, p [13] Belser LW, Mays L. Use of nitrifier activity measure ments to estimate the efficiency of viable nitrifier counts in soils and sediments. Appl Environ Microbiol 1982;43(4): [14] Gumaelius L, Smith EH, Dalhammar G. Potential biomarker for denitrification of wastewaters: effects of process variables and cadmium toxicity. Water Res 1996;3(12): [15] Kadlec RL, Knight RL. Treatment wetlands. Boca Raton, FL, USA: CRC Press-Lewis Publishers; [16] Verhoeven JTA. Nutrient dynamics in minerotrophic peat mires. Aquat Bot 1986;25(2): [17] Kansiime F, Nalubega M, van Bruggen JJA, Lijklema L. Variation of water quality in the Nakivubo wetland, Kampala-Uganda. Proceedings of Seventh International Conference on Wetland Systems for Water Pollution Control, November 11 16, Florida, 2. p [18] Cloete TE, Muyima NYO. Microbial community analysis: the key to the design of biological wastewater treatment systems. IAWQ Scientific and Technical Report No. 5. Cambridge: Cambridge University Press; pp. [19] Focht DD, Verstraete W. Biochemical ecology of nitrification and denitrification. Adv Microb Ecol 1977;1: [2] NEMA, The national environment (standards for discharge of effluent into water or on land) regulations, Uganda. [21] Lizhibowa M. Effects of pre-settled waste water on the growth of Cyperus papyrus,its performance on nutrient removal in Uganda. M.Sc. thesis, IHE, Delft, The Netherlands, pp. [22] Okurut TO, Rijs GBJ, van Bruggen JJA. Design and performance of experimental constructed wetlands in Uganda, planted with Cyperus papyrus and Phragmites mauritianus. Water Sci Technol 1999;4(3): [23] Eighmy TT, Bishop PL. Distribution and role of bacterial nitrifying populations in nitrogen removal in aquatic treatment systems. Water Res 1989;23(8): [24] Schreijer M, Kampf R, Toet S, Verhoeven J. The use of constructed wetlands to upgrade treated sewage effluents before discharge to natural surface water in Texel island, The Netherlands pilot study. Water Sci Technol 1997; 35(5): [25] Hunt PG, Poach ME. State of the art for animal wastewater treatment in constructed wetlands. Proceedings of the Seventh International Conference on Wetland Systems for Water Pollution and Control. International Water Association 2;1: [26] Johansson L. Use of light expanded clay aggregates (Leca) for removal of phosphorus from wastewater. Water Sci Technol 1997;35(5): [27] Reddy KR, D Angelo EM. Biogeochemical indicators to evaluate pollutant efficiency in constructed wetlands. Water Sci Technol 1997;35(5):1 1. [28] Armstrong J, Armstrong W. Phragmites australis a preliminary study of soil oxidising sites and internal gas transport pathways. New Phytol 1988;18: [29] Brix H. Functions of macrophytes in constructed wetlands. Water Sci Technol 1994;29(4):71 8. [3] Armstrong W, Armstrong J, Beckett PM. Measurement and modelling of oxygen release from roots of Phragmites australis. In: Cooper PF, Findlater BC, editors. Constructed wetlands in water pollution control. Oxford, UK: Pergamon Presss; 199. p [31] Eriksson PG, Weisner SEB. An experimental study on effects of submersed macropytes on nitrification and

11 J. Kyambadde et al. / Water Research 38 (24) denitrification in ammonium-rich aquatic systems. Limnol Oceanogr 1999;44(8): [32] Heidenwang I, Langheinrich U, Luderitz V. Self-purification in upland and lowland streams. Acta Hydrochim Hydrobiol 21;29(1): [33] K.orner S. Nitrifying and denitrifying bacteria in epiphytic communities of submerged macrophytes in a treated sewage channel. Acta Hydrochim Hydrobiol 1999;27(1): [34] Raff J, Hajek P-M. Zur Nitrifikation in FlieXgew.assern durch suspendierte und Nitrifikanten, 1. Mitteilung: Abh-.angigkeit der Nitrifikationsrate vom C-BSB und Ammoniumgehalt. GWF Gas Wasserfach: Wasser-Abwasser 1981;122:15 9.