ARTICLE IN PRESS

Water Research 38 (2004) 475–485

A comparative study of Cyperus papyrus and Miscanthidium

violaceum-based constructed wetlands for wastewater
treatment in a tropical climate
Joseph Kyambaddea,b, Frank Kansiimea, Lena Gumaeliusb,
Gunnel Dalhammarb,*
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-106 91 Stockholm, Sweden
Received 20 March 2003; received in revised form 22 September 2003; accepted 2 October 2003

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% ammonium-
nitrogen). No TRP removal was detected in control effluent. Nutrients (N and P) were significantly higher (po0:015) 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 30.7% P of the total N and P removed by treatment line 3 (Miscanthidium
violaceum). In addition, papyrus exhibited a significantly larger (p ¼ 0:000) number of adventitious roots than
Miscanthidium. Nitrifying bacteria attached to papyrus (2.15  10671.53  105 MPN/g DW) and Miscanthidium roots
(1.30  10478.83  102 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 2003 Elsevier Ltd. All rights reserved.

Keywords: Constructed wetlands; Cyperus papyrus; Miscanthidium violaceum; Nutrients; Tropical wetlands; Wastewater treatment

1. Introduction these wetlands are continuously being modified for

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.: +46-8-5537-8300; fax: 46-8-
5537-8483.
E-mail address: gunnel@biotech.kth.se (G. Dalhammar).
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

0043-1354/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2003.10.008
 ARTICLE IN PRESS
476 J. Kyambadde et al. / Water Research 38 (2004) 475–485
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,
2001 and August, 2002 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 con-
stant 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 2001 at
Makerere University botanical garden, Kampala, Ugan-
da. The design was a succession through a number of
reverse vertical flow containers 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 allow sufficient 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 0.58
and 0.82 m for CW1 (anaerobic), and 0.58 and 0.43 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/m2 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.
2.2. 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 0.064 m3/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 (NH4-N) was analyzed following
direct Nesslerization method [9]. A diazotization method
was used to analyze nitrite-nitrogen (NO2-N), while
nitrate-nitrogen (NO3-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-4000 spectrophotometer.
2.3. Measurement of plant biomass, nutrients and root
surface area
An overview of 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 examina-
tion, 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 10 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
 ARTICLE IN PRESS
J. Kyambadde et al. / Water Research 38 (2004) 475–485 477

Wastewater reservoir

1 1 2 2 3 3
CW1

CW2

CW3

CW4

CW5

CW6

Effluent

Wastewater reservoir

12mm gate valve

CW1
CW2
CW3
CW4 Effluent
CW5
CW6

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. [10]. 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.
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
ð1Þ
media used was according to Schmidt and Belser [12].
The tests were carried out in 10 ml culture tubes with
screw caps; the total test volume was 5.0 ml. One
milliliter of each 10-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
 ARTICLE IN PRESS
478 J. Kyambadde et al. / Water Research 38 (2004) 475–485

test media were included as negative controls. After
inoculation, the used root samples were oven dried at
103 C for dry weight determination.
2.5. Nitrification activity
Potential nitrification activity was carried out as
described by Belser and Mays [13] with slight modifica-
tions. Briefly, 10 g of root samples were incubated
aerobically at room temperature in 500 ml Erlenmeyer
flasks containing 300 ml of test media (1 g neutralized
bacteriological peptone (oxoid), 8.5 g NaCl, 0.095 g
ammonium sulfate, and 1.06 g sodium chlorate per
liter). For water and peat samples, 100 ml was added
to 200 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 10 ml
samples for nitrite every 45 min using Merck Spectro-
quant 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 nitrifica-
tion activity. Nitrite concentrations were calculated
from absorbance of samples and standard nitrite
(0.2 mg/l) using Beer–Lambert’s law.
2.6. Statistical analyses
Statistical analysis was performed with the package
MINITAB Release 13.31 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 physico-
chemical 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 (po0:005 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 7.770.1 8.170.0 8.370.1 8.470.1 8.370.1 8.270.1

EC (mS/cm) 796.3716.0 739.9 712.1 695.374.3 675.0729.2 642.8727.9 631.5733.1
Temp. ( C) 21.67 0.2 21.870.1 21.770.1 21.570.1 21.370.1 21.470.1
DO (mg/l) 0.3570.04 2.6470.29 2.6970.58 3.5870.98 3.8271.1 2.3070.68
NH4-N (mg/l) 28.970.3 26.771.6 26.771.2 26.271.7 23.071.4 20.870.3
NO2-N (mg/l) 0.2070.08 0.3770.02 0.0670.00 0.0670.01 0.0970.02 0.0770.01
NO3-N (mg/l) 0.2370.01 0.3470.01 0.3070.01 0.2870.01 0.2970.00 0.2870.00
TN (mg/l) 58.572.5 52.674.1 48.672.6 51.274.6 44.672.1 43.572.4
TRP (mg/l) 15.571.1 14.271.2 14.571.5 16.971.4 16.071.3 17.171.3

2 pH 7.770.1 8.070.0 6.870.0 6.870.0 6.670.0 6.770.0

EC (mS/cm) 796.3716.0 738710.7 457.37 13.5 386.178.3 325.674.9 29872.7
Temp. ( C) 21.670.0 21.670.4 22.270.2 22.070.3 21.870.2 21.770.1
DO (mg/l) 0.3570.04 2.5770.42 0.9870.24 0.5370.05 0.5870.08 0.7670.13
NH4-N (mg/l) 28.970.3 26.770.9 16.171.0 11.370.2 8.770.6 7.171.1
NO2-N (mg/l) 0.2070.08 0.3570.02 0.0970.03 0.0670.01 0.0270.00 0.0270.00
NO3-N (mg/l) 0.2370.01 0.3570.02 0.1670.01 0.1370.00 0.1070.01 0.0970.03
TN (mg/l) 58.572.5 53.073.8 26.372.6 25.271.2 15.571.2 16.171.1
TRP (mg/l) 15.571.1 14.571.2 6.370.6 4.870.2 3.370.3 2.671.1

3 pH 7.770.1 8.370.1 7.170.0 7.170.0 7.070.0 7.170.0

EC (mS/cm) 796.3716.0 740714.4 510.876.5 470.8712.1 416.5718.9 407.3729.7
Temp. ( C) 21.670.0 22.070.5 22.270.3 22.170.2 22.070.1 22.270.1
DO (mg/l) 0.3570.04 2.5270.32 0.5870.05 0.570.08 0.770.11 0.9170.2
NH4-N (mg/l) 28.970.3 26.771.5 16.970.5 15.370.7 12.471.1 11.171.7
NO2-N (mg/l) 0.2070.08 0.3670.01 0.0770.02 0.0770.02 0.0570.01 0.0470.00
NO3-N (mg/l) 0.2370.01 0.3470.01 0.1470.01 0.1370.01 0.1170.01 0.0770.01
TN (mg/l) 58.572.5 52.073.9 30.572.5 24.471.2 19.471.2 17.972.2
TRP (mg/l) 15.571.1 14.870.4 10.471.0 10.171.1 8.771.1 8.071.1
 ARTICLE IN PRESS
J. Kyambadde et al. / Water Research 38 (2004) 475–485 479

all variables). Water pH was significantly lower in
papyrus (p ¼ 0:001) than Miscanthidium (p ¼ 0:006)
CWs compared to the controls. Similarly conductivity
was lower in papyrus (p ¼ 0:02) than Miscanthidium
(p ¼ 0:03) CWs, whereas DO was lower in Miscanthi-
dium (p ¼ 0:003) than papyrus (p ¼ 0:004) CWs. Multi-
ple comparisons detected higher water temperature in
Miscanthidium (p ¼ 0:001) than papyrus (p ¼ 0:054).
The longitudinal variation of the average concentra-
tions of ammonium-nitrogen (NH4-N), nitrite-nitrogen
(NO2-N), nitrate-nitrogen (NO3-N), total-nitrogen (TN)
and total reactive phosphorus (TRP) is presented in
Table 1. The differences in concentrations of NH4-N,
NO3-N, TN and TRP between the three treatments were
highly significant (po0:004 for all variables). In contrast
no significant differences (p ¼ 0:281) in NO2-N concen-
trations were detected between treatments. In all
treatments, nitrite-nitrogen and nitrate-nitrogen were
detected but only in small concentrations. Multiple
comparisons showed significantly higher NH4-N re-
moval in papyrus (p ¼ 0:02) than Miscanthidium
(p ¼ 0:026) CWs compared to unplanted controls.
On the other hand, NO3-N was significantly higher
in unplanted controls than papyrus (p ¼ 0:027) and
No. of new main roots recruited
Miscanthidium (p ¼ 0:02) CWs. However, multiple
comparisons detected significantly high TN removal in
both papyrus (p ¼ 0:018) and Miscanthidium (p ¼ 0:017)
CWs that was nearly equal. Similarly, the longitudinal
TRP removal was significantly higher in papyrus
(p ¼ 0:003) than Miscanthidium (p ¼ 0:004) CWs in
comparison with the unplanted controls.
3.2. 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 0:072) 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 0:014) between the two plant species. The average
plant weight increment was 0.228 and 0.039 kg per CW
unit for papyrus and Miscanthidium, respectively.

500
450
400
350
per CW unit

300
250
200
150
100
50
0
0 1 2 3 4 5 6 7
Period (weeks)
Papyrus Miscanthidium

60
No. of new shoots per CW unit

50

40

30

20

10

0
wk0 wk1 wk2 wk3 wk4 wk5 wk6 wk7 wk8 wk9 wk10
Period (weeks)

Papyrus Miscanthidium

Fig. 2. Average weekly recruitment of roots and shoots per CW planted with Cyperus papyrus and Miscanthidium violaceum.
 ARTICLE IN PRESS
480 J. Kyambadde et al. / Water Research 38 (2004) 475–485

Though shoot recruitment rates were higher in papyrus canthidium. The mean radii of the main root of both
than Miscanthidium CWs (5 and 2 shoots per week), plant species did not vary significantly (p ¼ 0:196). In
respectively, one-way ANOVA did not detect significant contrast, the average length of the main root was
differences between the two plant species (p ¼ 0:14). significantly greater (p ¼ 0:03) in Miscanthidium than in
papyrus.

3.3. Estimation of root surface area of plants

3.4. Nitrifying bacteria
Results of root surface area determinations are shown
in Table 2. Papyrus exhibited a significantly larger The distribution of nitrifying bacteria in the different
number of adventitious roots (p ¼ 0:000) than Mis- layers of the constructed wetlands is depicted in Fig. 4.

3
Weight increment (kg) per CW

2
2.5 y = 0.0225x - 0.0372x
2
R = 0.9834
2
unit

1.5

1 2
y = 0.0015x + 0.0165x
2
0.5 R = 0.914

0
wk0 wk1 wk2 wk3 wk4 wk5 wk6 wk7 wk8 wk9 wk10
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) 24.6570.10 29.5570.74

Mean length of adventitious roots (cm) 2.2070.03 1.4870.03
Mean number of adventitious roots/main root 27777 5373
Mean radius of main root (cm) 0.2170.01 0.2370.02
Mean radius of adventitious roots (cm) 0.04670.00 0.06070.01
Average root surface area (cm2)a 208.6 72.2
a
Calculated using Eq. (1).
Log number (MPN/100 ml or gDW)

1.00E+08
1.00E +07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
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).
 ARTICLE IN PRESS
J. Kyambadde et al. / Water Research 38 (2004) 475–485 481

Numbers of ammonia oxidizing bacteria followed the and Miscanthidium were 0.0310, 0.0314, 0.147 and
pattern peat>papyrus>Miscanthidium>water column. 0.146 mg NO2/l/h.
The mean numbers of nitrifying bacteria in the papyrus
root mat (2.15  10671.53  105 MPN/g DW) were 3.6. Nitrogen and phosphorus content in plant tissues
significantly higher (p ¼ 0:000) than those asso-
ciated with the Miscanthidium root mat (1.30  104 Results of nutrient content in plant tissues are
78.83  102 MPN/g DW). depicted in Fig. 6. Concentrations of N were signifi-
cantly higher than P in both papyrus (p ¼ 0:037) and
Miscanthidium (p ¼ 0:004) tissues. In addition, N levels
3.5. Potential nitrification activity were significantly higher (p ¼ 0:014) in papyrus than in
Miscanthidium. Similarly, P levels were significantly
Results of potential microbial nitrification activities higher (p ¼ 0:04) in papyrus than in Miscanthidium.
for water, peat and root mat samples of papyrus and
Miscanthidium are depicted in Figs. 5a and b. Higher

N and P content (g/kg dry weight)

nitrification potential was found to be associated with
20
papyrus roots than Miscanthidium, water or peat 18
16
samples (Fig. 5a). The root mat samples of the two 14
12 Nitrogen
plant species showed similar nitrification capacities for 10
8 Phosphorus
the first 3 h. However, papyrus surpassed to attain a 6
4
maximum nitrite production of 0.8 mg/l in the next 1.5 h. 2
0
In contrast, Miscanthidium root samples attained a

t
ot

oo
o

oo
ro
ro

sh

sh
maximum of 0.514 mg/l in the first 3 h before it leveled s

um
ru

um
ru
py

di

py

di
hi
off (Fig. 5b). Similarly, high activity was detected in the
Pa

hi
nt

Pa

nt
ca

ca
peat although it was not significantly different from that is

is
M

M
of water (p ¼ 0:053). The activity for peat samples Plant tissue

reached a maximum of 0.213 mg nitrate-N/l in 7 h as Fig. 6. Nitrogen and phosphorus content in root and shoot
opposed to a maximum of 0.183 mg nitrate-N/l in the tissues of Cyperus papyrus and Miscanthidium violaceum during
water column samples after 5.5 h (Fig. 5b). The slopes of the 4th and 5th months of analysis. Bars indicate standard error
linear regression for water, peat and roots of papyrus of the mean (n ¼ 3).
Nitrite-N concentration (mg/l)
Nitrite-N concentration (mg/l)

0.25 0.25 y = 0.0309x

R2 = 0.9886
0.2 0.2
y = 0.0299x
0.15 0.15 R2 = 0.9816

0.1 0.1

0.05 0.05

0 0
0 2 4 6 8 10 12 0 1 2 3 4 5 6 7 8
Time (h) Time (h)

Nitrite-water Nitrite-Peat Nitrite-water Nitrite-Peat

0.9 y = 0.0027x
Nitrite-N concentration (mg/l)
Nitrite-N concentration (mg/l)

0. 9 2
0.8 R = 0.9649
0. 8
0.7
0. 7
0. 6 0.6
y = 0.0025x
0. 5 0.5 2
R = 0.9798
0.4
0. 4
0. 3
0.3
0. 2 0.2
0.1
0. 1
0 0
0 100 200 300 400 500 600 700 0 50 100 150 200 250 300 350
(a) Time (min) (b) 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.
 ARTICLE IN PRESS
482 J. Kyambadde et al. / Water Research 38 (2004) 475–485
4. 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 4–9.5 for the survival of most
bacteria [15]. The water pH value was slightly acidic (pH
6.6–6.8) in CWs planted with papyrus compared to
Miscanthidium (pH 7.0–7.1; 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 5.0 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 correspond-
ing planted wetland units and this was attributed to
algal photosynthesis. Effective nitrification has been
reported in systems with DO content of only 0.5 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 flow through of
nitrificants. In contrast, concentrations of DO decreased
in all vegetated CWs due to aerobic decomposition of
plant materials, nitrification and minimal surface aera-
tion resulting from vegetation coverage. The low nitrate
levels in planted CWs can be explained by heterotrophic
competition with nitrificants for oxygen [19], denitrifica-
tion and plant uptake. The values of electrical con-
ductivity and the concentrations of NH4-N, NO2-N,
NO3-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 below the Uganda
regulatory discharge limit of 10 mg/l [20] were obtained
in both planted CWs, with papyrus showing much lower
concentrations of up to 2.6 mg/l. However, the effluent
NH4-N concentration below the national discharge limit
of 10 mg/l could only be obtained in papyrus CWs. The
longitudinal NH4-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% NH4-N removal and 10.3% TRP accumula-
tion 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] (70% NH4-N) in bucket experiments at Kiriinya [22]
(o20% for both NH4-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 deni-
trification 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 substra-
tum surface containing free Ca2+, Fe2+, Mg2+ and
Al3+ 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 30.7% 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–denitrifi-
cation 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 com-
ponents and uptake of nutrients. Indeed, this was
supported by the difference in root surface area
(Papyrus, 208.6 cm2 per main root compared to Mis-
canthidium, 72.2 cm2) 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/m2/day) reported
for Phragmites australis [30], papyrus CWs provided
 ARTICLE IN PRESS
J. Kyambadde et al. / Water Research 38 (2004) 475–485
6.3 g O2/m2/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 addi-
tion to providing attachment sites and diffusible
oxygen to the bacteria, root mats increase waste-
water residence time and retention of suspended
organic particles, which upon degradation avail nutri-
ents to bacteria and plants. In fact, more nitrifying
bacteria were associated with papyrus roots (2.15  106
71.53  105 MPN/g DW) compared to Miscanthidium
roots (1.30  10478.83  102 MPN/g DW) and water
column (2.93  10378.83  101 MPN/100 ml), suggest-
ing 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 (1.67  10773.33  105 MPN/100 ml) and epiphy-
ton were comparably higher than those reported by
.
Korner [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 provid-
ing attachment sites, diffusible oxygen from the aerial
parts, and substrates from trapped decomposing waste-
water components.
Nitrifiers in the epiphyton have been estimated to
have an equally high influence on total system nitrifica-
tion 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 hetero-
trophs. 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 (2.93  103+8.83  101 MPN/
100 ml) in the water column.
483
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 concen-
trations 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, knowl-
edge of structural development and recruitment rates of
roots and the general growth rate of the macrophyte in
question is crucial. This influences plant–microorgan-
isms–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 asso-
ciated with planted CWs compared to unplanted
controls. Papyrus CWs showed markedly higher nu-
trient removal efficiencies, with higher N and P levels in
plant tissues in comparison to Miscanthidium. Similarly,
more shoots were developed by papyrus than Mis-
canthidium 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 Biotechnol-
ogy Policy Development (BIO-EARN). Authors are
 ARTICLE IN PRESS
484 J. Kyambadde et al. / Water Research 38 (2004) 475–485
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 do-
mestic wastewater. Hydrobiol Bull 1987;21(2):167–70.
[2] Azza NGT, Kansiime F, Nalubega M, Denny P.
Differential permeability of papyrus and Miscanthidium
root mats in Nakivubo swamp, Uganda. Aquat Bot
2000;67:169–78.
[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 Pro-
gramme, Wetlands Inspectorate Division, Ministry of
Water, Land and Environment, Kampala, 1998. p. 1–30.
[4] Gersberg RM, Elkins BV, Lyon SR, Golman CR. Role of
aquatic plants in wastewater treatment by artificial wet-
lands. Water Res 1986;20(3):363–8.
[5] Reddy KR, Patrick WH, Lindau CW. Nitrification–
denitrification at the plant root–sediment interface in
wetlands. Limnol Oceanogr 1989;34(6):1004–13.
[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;
1999. 300pp.
[7] Kansiime F, van Bruggen JJA. Distribution and
retention of faecal coliforms in the Nakivubo wetland in
Kampala, Uganda. Water Sci Technol 2001;44(11/12):
199–206.
[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 2002;27(1):
11–20.
[9] American Public Health Association (APHA). Standard
methods for the examination of water and waste water,
18th ed. Washington, DC, USA: APHA–AWWWA–
WEF; 1992.
[10] 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):239–48.
[11] Mutulewich VA, Strom PE, Finstein MS. Length of
incubation for enumerating nitrifying bacteria present in
various environments. Appl Microbiol 1975;29:265–8.
[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 Mono-
graph, vol. 9, 1982. p. 1027–42.
[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):
945–8.
[14] Gumaelius L, Smith EH, Dalhammar G. Potential
biomarker for denitrification of wastewaters: effects of
process variables and cadmium toxicity. Water Res
1996;30(12):3025–31.
[15] Kadlec RL, Knight RL. Treatment wetlands. Boca Raton,
FL, USA: CRC Press-Lewis Publishers; 1996.
[16] Verhoeven JTA. Nutrient dynamics in minerotrophic peat
mires. Aquat Bot 1986;25(2):117–38.
[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, 2000. p. 727–34.
[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; 1997. 10pp.
[19] Focht DD, Verstraete W. Biochemical ecology of nitrifica-
tion and denitrification. Adv Microb Ecol 1977;1:135–214.
[20] NEMA, 1999. 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, 1995. 173pp.
[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;40(3):265–71.
[23] Eighmy TT, Bishop PL. Distribution and role of bacterial
nitrifying populations in nitrogen removal in aquatic
treatment systems. Water Res 1989;23(8):947–55.
[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):231–7.
[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 2000;1:29–36.
[26] Johansson L. Use of light expanded clay aggregates (Leca)
for removal of phosphorus from wastewater. Water Sci
Technol 1997;35(5):87–93.
[27] Reddy KR, D’Angelo EM. Biogeochemical indicators to
evaluate pollutant efficiency in constructed wetlands.
Water Sci Technol 1997;35(5):1–10.
[28] Armstrong J, Armstrong W. Phragmites australis—a
preliminary study of soil oxidising sites and internal gas
transport pathways. New Phytol 1988;108:373–82.
[29] Brix H. Functions of macrophytes in constructed wetlands.
Water Sci Technol 1994;29(4):71–8.
[30] Armstrong W, Armstrong J, Beckett PM. Measurement
and modelling of oxygen release from roots of Phragmites
australis. In: Cooper PF, Findlater BC, editors. Con-
structed wetlands in water pollution control. Oxford, UK:
Pergamon Presss; 1990. p. 41–52.
[31] Eriksson PG, Weisner SEB. An experimental study on
effects of submersed macropytes on nitrification and
 ARTICLE IN PRESS
J. Kyambadde et al. / Water Research 38 (2004) 475–485 485

denitrification in ammonium-rich aquatic systems. Limnol
Oceanogr 1999;44(8):1993–9.
[32] Heidenwang I, Langheinrich U, Luderitz V. Self-purifica-
tion in upland and lowland streams. Acta Hydrochim
Hydrobiol 2001;29(1):22–33.
.
[33] Korner S. Nitrifying and denitrifying bacteria in epiphytic
communities of submerged macrophytes in a treated
sewage channel. Acta Hydrochim Hydrobiol 1999;27(1):
27–31.
[34] Raff J, Hajek P-M. Zur Nitrifikation in FlieXgew.assern
durch suspendierte und Nitrifikanten, 1. Mitteilung: Abh-
a. ngigkeit der Nitrifikationsrate vom C-BSB und Ammo-
niumgehalt. GWF Gas Wasserfach: Wasser-Abwasser
1981;122:15–9.