Ecological Engineering 102 (2017) 112126

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Wastewater remediation by optimum dissolve oxygen enhanced by

macrophytes in constructed wetlands
Faiza Rehman, Arshid Pervez , Qaisar Mahmood , Bahadar Nawab
Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: As new ways are being sought to treat wastewater in natural and constructed wetlands (CWs), the role of
Received 6 September 2016 oxygen released by the macrophytes for degrading organic waste has inordinate signicance. The current
Received in revised form 11 January 2017 investigation assessed the optimum dissolve oxygen (DO) added by Typha latifolia and Phragmites aus-
Accepted 29 January 2017
tralis when exposed to the optimum temperature and light intensity. A wireless network system (WSN)
monitored the environmental conditions which was designed and deployed at the experimentation site.
Keywords:
The plants were exposed to 16 different combinations of temperature and light intensity. The optimum
Dissolve oxygen
DO in the rhizosphere of T. latifolia was recorded when plants were exposed to 30 C and 35 C implying
Removal efciency
Constructed wetlands
that the optimum temperature range was 3035 C with 15 000 lx light intensity. The optimum DO in the
rhizosphere of P. australis was recorded when plants were exposed to the 35 C temperature and 10 000 lx
to 15 000 lx light intensity. A strong correlation was found between DO, total chlorophyll and fresh plant
biomass at the optimum temperature and light intensity combinations in both plants. The maximum DO
in the substratum of CWs proved to play a signicant role in reducing hydraulic retention time (HRT) in
vegetated CWs.
2017 Elsevier B.V. All rights reserved.

1. Introduction the roots of Potamogeton perfoliatus (submerged macrophytes) was

With the advent of industrialization and use of chemicals for
domestic purposes, agriculture and pharmaceutical products, the
conventional treatment systems are unable to abate the environ-
mental pollution (Hayder et al., 2015). CWs have the potential to
achieve the required treatment goals at low cost and energy utiliza-
tion as compared to the conventional treatment systems (Mthembu
et al., 2013). However, old fashioned CWs would not be effective for
the treatment of large volume of wastewater containing emergent
pollutants. In CWs, macrophytes play a vital role in wastewater
treatment through nutrients, salts and heavy metals uptake and
release of oxygen in rhizosphere (Vymazal, 2011; Wenlin et al.,
2014). The oxygen release by plants in the rhizosphere augments
degradation of organic matter in wastewater. However, this oxy-
gen might become a rate limiting factor in constructed wetlands
(Brix, 1994; Uteau et al., 2015). Some scientists demonstrated that
a part of the oxygen produced as a byproduct during photosyn-
thesis is transported to the aerenchyma cells which are a structural
adaptation of the aquatic macrophytes (Connel et al., 1999; Headley
and Tanner, 2008; Zhang et al., 2014). The oxygen released from
Corresponding author.
E-mail addresses: pervez@ciit.net.pk (A. Pervez), mahmoodzju@gmail.com
(Q. Mahmood).
directly proportional to the rate of photosynthesis in plant shoot.
Thus when the amount of O2 in the aerenchyma cells and root
region surpasses the plant demand, diffusion may occur into plant
rhizosphere (Armstrong, 1979; Brix, 1997). However, it is also
believed that a signicant amount of oxygen is also transported
from the atmosphere to the rhizosphere through macrophytes e.g.
in Nuphar lutea in which oxygen entered the youngest emerged
leaves due to gas pressure gradient and entered into the petioles
and large blades of older leaves (Armstrong and Armstrong, 1988;
Konnerup et al., 2011). Moreover, the direct diffusion of oxygen
was observed in emergent plants with cylindrical culms and linear
leaves e.g. P. australis (Pedersen et al., 2004; Afreen et al., 2007).
T. latifolia and P. australis are the emergent macrophytes having
an effective aeration system and large intercellular spaces meant
for oxygen within plant to accomplish the respiratory demand
of submerged tissues and rhizosphere. Additionally, the ability of
reeds to transport oxygen to the rhizosphere is proved to be a
crucial mechanism in the removal of BOD and nitrogen (Lavrova
and Koumanova, 2013). This oxygen release from the roots of
macrophytes is affected by physical factors. It can be maximized to
enhance the pollutant removal in CW using different macrophytes
under same climatic conditions, types of wastewater, substrate
type and same species of macrophytes under different climatic con-
ditions. Large differences were found in efciency of same species
to remove one or more types of pollutants and the selection of

http://dx.doi.org/10.1016/j.ecoleng.2017.01.030
0925-8574/ 2017 Elsevier B.V. All rights reserved.
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126 113

Fig. 1. (A) Design of a vertical ow constructed wetland with two types of gravel used as substratum and a perforated PVC pipe inserted in the center for measuring dissolved
oxygen. (B) The growth chamber with deployed wireless sensor network having three sensor nodes, a data collecting and processing point, two communication technologies
and a display system. (C) Photograph of the growth chamber with deployed wireless sensor network. Three nodes are composed of temperature, humidity and light intensity
sensors.

Table 1 Table 2
Composition of synthetic wastewater. Components of nutrient solution.

Synthetic inuent Concentration Components (g/L) Components (g/L) Components (g/L)

Analytical grade glucose As per requirement Na2 -EDTA 5 FeCl2 4H2 O 3.58 CoCl2 6H2 O 0.5
NaHCO3 1 g/L NaOH 11 MnCl2 2H2 O 2.5 (NH4 )6 Mo7 O24 4H2 O 0.5
Trace element solution 1 ml/L CaCl2 2H2 O 7.34 ZnCl2 1.06 CuCl2 2H2 O 0.14
MgCl2 1 g/L
KH2 PO4 1 g/L
(NH4 )2 SO4 0.24 g/L

environmental conditions within the greenhouses and elds to

macrophyte species is sometimes important for the removal of cer-
tain pollutant under specic environmental conditions (Wiebner
et al., 2002; Brisson and Chazarenc, 2008). Similarly the growth
stages of different macrophytes also affect the release of oxygen. In
P. australis the oxygen released during budding, elongation, mat-
uration, and dormancy phases was three times higher than the
oxygen released during the growth stages of other plants (Zhang
et al., 2014).
Scientists are working on automated systems also called
Wireless Sensor Networks (WSN) to monitor and control the
get the maximum possible process control. Small size and data
sensing accuracy, high sampling frequency, energy efcient bat-
tery powered compressed ready-to-deploy units make wireless
sensor enabled technologies ideal for environmental sensing appli-
cations. Multiple sensing nodes equipped with sensing elements
like temperature, light humidity, wind speed, gases concentra-
tion, soil/atmospheric moisture level etc. (Akshay et al., 2012). The
present study aimed to investigate the optimum DO levels added
by the P. australis and T. latifolia to the substratum of the CW under
controlled temperature and light intensity combinations. Further-
114 F. Rehman et al. / Ecological Engineering 102 (2017) 112126
more, wireless sensor network system was used to dene the effect
of DO on organic matter removal in synthetic wastewater.
2. Materials and methods
2.1. Plant sampling and design of constructed wetlands
P. australis and T. latifolia were collected from a natural wet-
land. The roots were washed with tap water and planted in green
house to produce uniform sized plants for the experiment. The
uniform sized plants were planted in constructed wetlands (Soda
et al., 2007). Vertical ow constructed wetlands (VFCWs) were con-
structed in cylindrical plastic buckets with 44 cm diameter and
80 cm height with an outlet at the bottom. Large gravels (3 cm)
were placed at the bottom up to 20 cm above which a layer of 30 cm
small gravels (pebble size 1 cm) were placed as substrate (Lavrova
and Koumanova, 2013). A 5 cm wide and 100 cm long PVC pipe
with 0.5 cm wide perforations (to allow water to seep through) on
the lower side (up to 50 cm) were xed in the center of the gravel
layers. In order to prevent atmospheric oxygen to diffuse in the
substratum, the buckets were plugged with Styrofoam and wax
(Fig. 1A). Constructed wetlands (with 12 monoculture plants each)
were placed under controlled temperature and light intensity in a
growth chamber. Florescent bulbs were used to obtain light of dif-
ferent intensities. For temperature maintenance automatic heater
with a blower was used (Fig. 1B).
2.2. Wastewater composition and characterization
Synthetic wastewater was used for the study. The components
of wastewater are summarized in Tables 1 and 2. For COD and
BOD analytical grade glucose was used. 1 g of glucose was added
to make 1 g/L COD approximately (Verstraete, 2005). The wastew-
ater was also supplemented with the nutrients required for the
plants (Johnston, 2004). The inuent wastewater characteristics are
summarized in Table 3.
Fig. 2. Block diagram of Wireless Network System.
Table 3
Characteristics (approximate levels) of pretreatment wastewater.
Parameters Concentration
COD 600680 mg/L
BOD 380 mg/L
pH 6.5
Temperature 25.6 C
2.3. Monitoring of controlled temperature and light intensity
with Wireless Sensor Network (WSN)
For the measurement of temperature, light intensity and humid-
ity, a specially designed wireless sensor network system was
deployed in the growth chamber. The system consisted of three
sensor nodes, a receiver hub; database and web interface (Fig. 2).
The sensor nodes were equipped with temperature, humidity (DHT
11) and light intensity (LDR) sensors. The communication between
the sensor nodes and the receiver station was achieved via 2.4 GHz
RF transmitter and receiver wireless modules. The data were col-
lected and displayed through a website at ten minutes interval.
The sensor nodes were installed inside a greenhouse having air
temperature, humidity and leaf temperature to collect the relative
data (Park and Park, 2011). Similarly a wireless monitoring and
control system was used for actual time data collection, reduced
human intervention and to avoid complex wiring system (Dhumal
and Chitode, 2013; Abinayaa and Jayan, 2014).
For verication light intensity and temperature were measured
with the help of a digital lux meter (LX-1010B) and a thermometer
(HTC-8) respectively.
2.4. Experimental design
The VFCWs were initially established at Research Nursery
Farm, COMSATS Institute of Information Technology (CIIT) Abbot-
tabad Pakistan. Plants were initially fed with Hoagland solution
(Ashton et al., 2011). Wastewater irrigation was started when
the mean height of T. latifolia and P. australis was on average
25 cm and 32 cm respectively. Triplicate units with each plant
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126
Fig. 3. DO recorded day to day from the substratum of non-vegetated and vegetated CWs with T. latifolia and P. asutralis. (a) 25 C5000 lx; (b) 25 C10 000 lx; (c)
25 C15 000 lx; (d) 25 C20 000 lx. Different alphabets show signicant difference between the maximum DO recorded from the rhizosphere of vegetated and non-vegetated
CWs.
were placed under controlled temperature and light intensity.
Three replicates of non-vegetated VFCWs were placed as con-
trolled units. Sixteen combinations of variable temperature and
light intensity i.e. 25 C5000 lx; 25 C10 000 lx; 25 C15 000 lx;
25 C20 000 lx; 30 C5000 lx; 30 C10 000 lx; 30 C15 000 lx;
30 C20 000 lx; 35 C5000 lx; 35 C10 000 lx; 35 C15 000 lx;
35 C20 000 lx; 40 C5000 lx; 40 C10 000 lx; 40 C15 000 lx;
40 C20 000 lx were used for the experiment.
The experiment started with the mean size for P. australis 30 cm
and T. latifolia 25 cm. The systems were exposed to each tempera-
ture and light intensity combination till optimum release of oxygen
by the plants to the substratum of the wetlands. Before experiment
was started the dissolved oxygen in the wastewater was reduced
to zero by nitrogen purging method through the perforated pipe
inserted in the center of the substratum.
Fresh plant biomass was determined before and after (18 days)
experiment. However in order to avoid any trauma and effect on
ongoing growth rate of the macrophytes, plants of same size and
fresh biomass were established in backup CWs for measuring fresh
plant biomass during the experimentation. Oxygen released in the
rhizosphere was measured after single hour for 8 h daily. BOD, COD
and chlorophyll content were measured after every 24 h.
115
2.5. Analytical procedures
Efuent samples were analyzed for DO, BOD, COD, Electric Con-
ductivity (EC), pH, and turbidity on daily basis. DO meter (Model
no. NeoMet DO-300L) was used to measure the concentration of
DO from the substratum of CWs through perforated PVC pipe. Five
days BOD5 standard test using DO meter and Winkler bottles was
used to measure the BOD concentration from efuent samples. COD
was measured by using closed reux colorimetric method (APHA,
1998). EC, pH were measured with the multi parameters instru-
ment (Jenway model 520). Turbidity was measured with turbidity
meter (Eutech, TN-100). Total chlorophyll was measured with the
atLEAF digital chlorophyll meter.
The % removal efciencies of wetlands were calculated accord-
ing to the following equation
 Cf

% Removal = Ci 100
Ci
where Ci is the initial concentration (mg/L) of any parameter
in inuent wastewater and Cf is the nal concentration (mg/L) of
that specic parameter in efuent samples. To make more clear
comparisons removal efciencies achieved by CWs and hydraulic
116 F. Rehman et al. / Ecological Engineering 102 (2017) 112126

Fig. 4. DO recorded day to day from the substratum of non-vegetated and vegetated CWs with T. latifolia and P. asutralis, (a) 30 C5000 lx; (b) 30 C10 000 lx; (c)
30 C15 000 lx; (d) 30 C20 000 lx. Different numbers indicate the signicant difference between the DO recorded from the substratum of vegetated and non-vegetated
CWs.

retention times were determined at 16 different temperature and ance (ANOVA) was used for fresh plant biomass of both plants
light intensity combinations (Table 5). separately at 16 different temperature and light intensity combi-
nations. The correlation among DO, total chlorophyll, fresh plant
biomass, COD and BOD was determined using Microsoft excel 10.
2.6. Statistical analysis

A two-way analysis of variance (ANOVA) using SigmaPlot was

used for DO released by T. latifolia and P. australis in substratum of
vegetated and non-vegetated CWs and one-way analysis of vari-

Table 4
The mean pH and water temperature values of efuent samples recorded after 18 days experiment from CWs with T. latifolia, P. australis and without vegetation.

Temperature and light CWs with T. latifolia CWs with P. australis Non-vegetated CWs
intensity combinations

pH Temperature ( C) pH Temperature ( C) pH Temperature ( C)

25 C5000 lx 6.38 0.05 25.5 C 0.945 6.15 0.05 26 C 0.914 6.29 0.05 27.4 C 0.825
25 C10 000 lx 6.18 0.09 26.4 C 0.847 6.29 0.01 26.7 C 0.822 6.00 0.07 25.3 C 0.903
25 C15 000 lx 6.36 0.02 26.2 C 0.917 6.43 0.05 26.3 C 0.775 6.18 0.03 25.7 C 0.825
25C-20000 lx 6.51 0.04 25 C 0.902 6.15 0.05 25 C 0.785 6.26 0.07 25.7 C 0.721
30 C5000 lx 6.58 0.01 26.5 C 0.795 6.45 0.01 26.1 C 0.882 6.09 0.05 26.7 C 0.698
30 C10 000 lx 6.25 0.05 26.8 C 0.783 6.21 0.07 26.4 C 0.941 6.25 0.07 25.8 C 0.745
30 C15 000 lx 6.31 0.06 27.6 C 0.762 6.19 0.02 27 C 0.909 6.18 0.04 26.1 C 0.723
30 C20000 lx 6.15 0.05 27.8 C 0.900 6.40 0.06 25.5 C 0.645 6.25 0.04 26 C 0.940
35 C5000 lx 6.18 0.04 26.6 C 0.789 6.55 0.04 26 C 0.731 6.24 0.08 25.4 C 0.675
35 C10 000 lx 6.58 0.01 27.5 C 0.905 6.23 0.05 26.4 C 0.945 6.18 0.01 25.7 C 0.941
35 C15 000 lx 6.28 0.02 26.5 C 0.811 6.25 0.01 27.1 C 0.945 6.10 0.03 25.6 C 0.742
35 C-20000 lx 6.45 0.04 26.7 C 0.760 6.41 0.04 26 C 0.945 6.15 0.05 26.4 C 0.801
40 C5000 lx 6.50 0.07 27.8 C 0.679 6.27 0.06 25.5 C 0.945 6.45 0.04 27.4 C 0.735
40 C10 000 lx 6.12 0.03 27.6 C 0.781 6.29 0.03 25.7 C 0.945 6.28 0.05 27 C 0.801
40 C15 000 lx 6.35 0.05 26.9 C 0.810 6.50 0.05 25.6 C 0.945 6.21 0.04 26.6 C 0.931
40 C20 000 lx 6.21 0.06 26.9 C 0.671 6.45 0.05 26.7 C 0.945 6.28 0.07 25.8 C 0.922
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126 117

Fig. 5. DO recorded day to day from the substratum of non-vegetated and vegetated CWs with T. latifolia and P. asutralis. (a) 35 C5000 lx; (b) 35 C10 000 lx; (c)
35 C15 000 lx; (d) 35 C20 000 lx. Different alphabets indicate signicant difference between maximum.

3. Results and discussion
The values of pH and temperature from the efuent of all CWs
recorded on day to day basis were not signicantly different from
each other. However mean pH, and water temperature values of
the efuent samples after 18 days experiment from vegetated and
non-vegetated CWs when exposed to 16 variable temperature and
light intensity combinations are mentioned in Table 4.
3.1. Effect of temperature and light intensity combination on DO
with P. australis it was 0.05 0.005 mg/L. In non-vegetated CW it
remained between 0 to 0.01 0.007 mg/L (Fig. 3b). Moreover the
maximum DO value measured in the rhizosphere of T. latifolia and
P. australis when exposed to 25 C15 000 lx was 0.08 0.005 mg/L
and 0.09 0.005 mg/L respectively. In non-vegetated CW it did not
exceed from 0.01 0.007 mg/L (Fig. 3c). However the maximum
DO recorded in the rhizosphere of T. latifolia when exposed to
25 C20 000 lx was 0.03 mg/L whereas in the rhizosphere of P. aus-
tralis the DO never increased from 0.04 mg/L. In non-vegetated CWs
the DO did not increase from 0.01 mg/L (Fig. 3d).

3.1.1. Effect of 25 C5000 lx; 25 C10 000 lx; 25 C15 000 lx;
25 C20 000 on DO; 25 C20 000 lx
The maximum DO recorded in the rhizosphere of T. latifolia
when exposed to 25 C5000 lx was 0.06 0.004 mg/L and P. aus-
tralis was 0.05 0.005 mg/L. In non-vegetated CW, the level of
DO did not exceed from 0.01 0.005 mg/L (Fig. 3a). Similarly the
highest DO recorded from the substratum of CWs with T. lati-
folia when exposed to 25 C10 000 lx was 0.11 0.005 mg/L and
3.1.2. Effect of 30 C5000 lx; 30 C10 000 lx; 30 C15 000 lx;
30 C20 000 lx on DO
The maximum value of DO measured in the rhizosphere of T. lat-
ifolia when exposed to 30 C5000 lx was 0.08 0.008 mg/L and of
P. australis was recorded 0.07 0.009 mg/L. In non-vegetated CW it
remained between 0 0.003 mg/L and 0.01 0.005 mg/L (Fig. 4a).
Similarly the highest value of DO recorded from the rhizosphere of
T. latifolia when exposed to 30 C10 000 lx was 0.56 0.014 mg/L
118 F. Rehman et al. / Ecological Engineering 102 (2017) 112126

Fig. 6. DO recorded day to day from the substratum of non-vegetated and vegetated CWs with T. latifolia and P. asutralis, (a) 40 C5000 lx; (b) 40 C10 000 lx; (c)
40 C15 000 lx; (d) 40 C20 000 lx. Same alphabets show no signicant difference between DO in the substratum of vegetated and non-vegetated CWs.

and of P. australis was 0.18 0.008 mg/L. In non-vegetated CW the
maximum DO recorded was 0.01 0.007 mg/L (Fig. 4b). Moreover
the highest DO was recorded from the rhizosphere of T.latifolia
when exposed to 30 C15 000 lx was 1.56 0.105 mg/L and P. aus-
tralis was 0.41 0.007 mg/L. In non-vegetated CW the value of
DO never exceeded from 0.01 0.005 mg/L (Fig. 4c). However the
maximum DO recorded from the rhizosphere of T. latifolia when
exposed to 30 C20 000 lx was 0.03 mg/L and of P. australis was
0.04 mg/L. Moreover the value of DO in the substratum of non-
vegetated CW remained between 0 and 0.01 mg/L (Fig. 4d).
3.1.3. Effect of 35 C5000 lx; 35 C10 000 lx; 35 C15 000 lx;
35 C20 000 lx on DO
When T. latifolia and P australis were exposed to 35 C5000 lx,
the highest value of DO recorded was 0.41 0.007 mg/L and
0.18 0.019 mg/L respectively. However in non-vegetated CW it
remained 0.01 0.005 mg/L (Fig. 5a). Similarly the maximum value
of DO recorded in the rhizosphere of T. latifolia and P. aus-
tralis when exposed to 35 C 10 000 lx was 0.95 0.014 mg/L
and 1.91 0.056 mg/L respectively . In non-vegetated CW it never
increased from 0.01 0.004 mg/L (Fig. 5b). Moreover the highest
value of DO recorded from the rhizosphere of T. latifolia when
exposed to 35 C15000 lx was 1.67 0.094 mg/L whereas in the
rhizosphere of P. australis it was 2.16 0.021 mg/L (Fig. 5c). How-
ever the maximum DO recorded from the rhizosphere of T. latifolia
when exposed to 35 C20 000 lx remained 0.03 0.007 mg/L dur-
ing the course of the experiment whereas in the rhizosphere of P.
australis it was 0.04 0.008 mg/L. Moreover the value of DO in the
substratum of non-vegetated CW remained between 0 0.005 and
0.01 mg/L (Fig. 5d).
3.1.4. Effect of 40 C5000 lx; 40 C10 000 lx; 40 C15 000 lx;
40 C20 000 lx on DO
The maximum value of DO in the rhizosphere of T. latifolia and
P. australis when exposed to 40 C5000 lx was 0.03 0.007 mg/L
and 0.04 0.008 mg/L respectively. However, the DO recorded
from the substratum of non-vegetated CWs remained between
0 0.005 and 0.01 0.005 mg/L (Fig. 6a). Similarly the highest value
of DO recorded from the rhizosphere of T. latifolia and P. australis
when exposed to 40 C10 000 lx was 0.04 0.008 mg/L. How-
ever DO in the substratum of non-vegetated CWs never increased
from 0.01 0.005 mg/L (Fig. 6b). Furthermore the maximum DO
in the rhizosphere of T. latifolia and P. australis when exposed to
40 C15 000 lx remained 0.04 0.007 mg/L. However the value of
DO in the substratum of non-vegetated CWs never increased from
0.01 0.006 mg/L (Fig. 6c). Similarly the maximum DO in the rhizo-
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126
Fig. 7. Optimum DO recorded from the rhizosphere of (a) T. latifolia and (b) P. australis at 16 different temperature ( C) and light intensity (lx) combinations. The mean values
of DO are plotted, different alphabetical letters show signicant difference among the optimum DO at variable combinations of temperature and light intensity.
sphere of T. latifolia and P. australis when exposed to 40 C20 000 lx
remained 0.04 0.008 mg/L with insignicant variations observed
on day to day basis. However the value of DO in the substra-
tum of non-vegetated CWs never increased from 0.01 0.007 mg/L
(Fig. 6d). The optimum DO recorded from the rhizosphere of T.
latifolia and P. asutralis at 16 different combinations are shown
in Fig. 3a and b respectively. Different letters indicate signicant
difference between DO values.
3.2. Relationship of temperature and light intensity combination
and DO in rhizosphere of T. latifolia
In vertical ow constructed wetlands, plants play vital role
to transport oxygen either produced by photosynthesis from
their leaves through stems and roots into the rhizosphere or by
direct conveyance from atmosphere to the substratum (Brix, 1994)
through an effective aeration system following the process of inter-
nal convective through-ow of gases (Armstrong and Armstrong,
1988; Bendix et al., 1994). A number of factors e.g. air temperature,
water temperature, wind speed, air pressure, humidity, oxygen
concentration in the surrounding environment, plant species, type
of substrate and redox state inuence the oxygen release rate from
the roots of plants (Stottmeister et al., 2003; Bezbaruah and Zhang,
2005; Konnerup et al., 2011).
The role of light intensity and temperature on rate of oxygen
release from the roots of aquatic macrophytes has been demon-
strated both in the eld and laboratory (Madsen and Sand-Jensen,
1994; Schwarz et al., 2000; Soda et al., 2007). The results of the
present study indicated that the release of oxygen from the roots
of T. latifolia was inuenced by the temperature and light inten-
sity (Fig. 7a). The temperature and light intensity combination of
35 C15 000 lx were highly effective in oxygen release from the
roots of the plants. The level of DO recorded from the substratum of
constructed wetlands with plants exposed to second highest tem-
perature i.e. 30 C and same light intensity showed no substantial
119
difference. However the continuous signicant decrease in the DO
in the substratum at 35 C and 10 000 lx and 35 C5000 lx speci-
es the dynamic role of light intensity difference in root release of
oxygen as proved by Tavechio and Thomas. Moreover keeping the
light intensity at 15 000 lx and lowering the temperature to 25 C
showed substantial difference in DO level in the rhizosphere of T.
latifolia, which indicates the forceful effect of temperature variance
from 30 C to 25 C on oxygen release.
Furthermore a major difference in the level of DO was observed
when temperature and light intensity were lowered together i.e.
at 30 C10 000 lx. When the DO at 30 C10 000 lx was compared
with the 35 C10 000 lx a considerable variance was found which
establishes the part of temperature in release of oxygen from the
roots of T. latifolia and that the difference in temperature between
35 C and 30 C became dominant at lower light intensity. These
conclusions are analogous to the claim that oxygen released by the
roots of macrophytes is greater in high light intensity and high tem-
perature and is lowered when light intensity and temperature are
decreased (Dong et al., 2011)
Likewise it was observed that the combinations having tem-
peratures 25 C and 40 C and light intensity 20 000 lx were not
favorable for the release of oxygen from the roots of T. latifolia. The
outcomes of the current study are also concurrent with the results
found by Soda et al. (2007) which identied that the aquatic plants
have optimal values of temperature and light intensity for oxygen
release from their roots. The current study veried that the opti-
mal temperature range for T. latifolia to release maximum oxygen to
the rhizosphere lies between 30 C and 35 C whereas the optimum
light intensity determined was 15 000 lx.
3.3. Relationship of temperature and light intensity combination
and DO in rhizosphere of P. australis
The oxygen released from the roots of P. australis has been
studied under the effect of variable environmental conditions like
120 F. Rehman et al. / Ecological Engineering 102 (2017) 112126
Fig. 8. Initial and nal total chlorophyll content of (A) T. latifolia and (B) P. australis measured with the help of digital chlorophyll meter. Plants are exposed to 16 different
combinations of temperature and light intensity.
wind speed, temperature, type of substratum and humidity level
(Armstrong et al., 1992; Brix, 1994). Many researches proved that
among many other environmental factors temperature and light
intensity also play vital role in root oxygen release by P. australis
(Weibner et al., 2002; Sisikala et al., 2009). However the best possi-
ble combination of temperature and light intensity to affect the
optimum oxygen release from the roots of different species of
macrophytes was understudied. In our study the results indicated
the highest DO recorded at 35 C15 000 lx which was not sig-
nicantly different from DO recorded at 35 C10 000 lx (Fig. 7b).
However a signicantly low DO at 35 C5000 lx combination indi-
cated the active role of light intensity difference between 10 000 lx
and 5000 lx. The results are concurrent with the study conducted
by Armstrong et al. (1991) that the specic light intensity plays
major role in root oxygen release in P. australis as it is signicantly
slowed down during night and can be enhanced by controlling light
intensity required by the plant.
Likewise, the DO recorded at 30 C15 000 lx combination was
also substantially lower than the DO recorded at 35 C15 000 lx
and 35 C10 000 lx showing the inuence of temperature modi-
cation between 35 C and 30 C on P. australis. However, the value
was higher than the DO recorded at 35 C5000 lx verifying the
forceful role of light intensity difference between 10 000 lx and
5000 lx. It was found that the combinations with temperature 25 C
and light intensity 5000 lx did not affect the release of oxygen from
the roots of P. australis signicantly. However the temperature 40 C
and light intensity 20 000 lx was proved to be critical for the plants.
The plants turned yellowish gradually and nally deteriorated com-
pletely during the experimentation. Thus the study suggested that
an optimum temperature for P. australis to add maximum oxygen to
the substratum through its roots was 35 C, whereas an optimum
light intensity ranged between 10 000 lx to 15 000 lx. Moreover a
single unfavorable environmental factor can contribute effectively
to the release of oxygen from the roots of the macrophytes. Conse-
quently maintaining the favorable temperature and light intensity
combination for the macrophytes to release optimum oxygen from
their roots can help improve the efciency of the CWs.
3.4. Effect of temperature and light intensity combinations on
total chlorophyll of macrophytes
A gradual decrease in the total chlorophyll of T. latifolia from
0.074 mg/cm2 to 0.67 mg/cm2 whereas from 0.030 mg/cm2 to
0.021 mg/cm2 in P. australis was observed in 18 days experi-
ment when exposed to 25 C5000 lx. Similarly when plants were
exposed to 25 C10 000 lx the total chlorophyll decreased from
0.076 mg/cm2 to 0.072 mg/cm2 and 0.030 mg/cm2 to 0.026 mg/cm2
in T. latifolia and P. australis respectively . At 25 C15 000 lx the
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126
Fig. 9. Correlation of DO and total chlorophyll of T. latifolia (a) 30 C10 000 lx; (b) 30 C15 000 lx; (c) 35 C10 000 lx; (d) 35 C15 000 lx and P. australis (A) 30 C10 000 lx;
(B) 30 C15 000 lx; (C) 35 C10 000 lx; (D) 35 C15 000 lx. Mean values of DO and total chlorophyll represented are recorded on day to day basis.
total chlorophyll in T. latifolia stayed between 0.072 mg/cm2 and
0.071 mg/cm2 with no signicant variation however in P. australis
it decreased from 0.028 mg/cm2 to 0.025 mg/cm2 . When plants
were exposed to 25 C20 000 lx the total chlorophyll in T. latifo-
lia decreased from 0.075 mg/cm2 to 0.042 mg/cm2 . However in P.
australis it decreased from 0.030 mg/cm2 to 0 mg/cm2 in 18 days.
The plant was gradually turned yellowish and nally withered.
Similarly when plants were exposed to 30 C5000 lx the
total chlorophyll increased from 0.060 mg/cm2 to 0.071 mg/cm2
in 18 days. However in P. australis it gradually decreased
from 0.028 mg/cm2 to 0.024 mg/cm2 . Likewise when plants were
exposed to 30 C10 000 lx the chlorophyll increased from 0.063
to 0.068 mg/cm2 and from 0.027 mg/cm2 to 0.030 mg/cm2 in T.
latifolia and P. australis respectively. Similarly a gradual increase
in the total chlorophyll was observed from 0.064 mg/cm2 to
0.068 mg/cm2 and 0.028 mg/cm2 to 0.03 mg/cm2 in T. latifolia and P.
asutralis respectively in 11 days when exposed to 30 C15 000 lx.
Moreover when plants were exposed to 30 C20 000 lx the total
chlorophyll decreased from 0.068 to 0.028 mg/cm2 and from
0.030 mg/cm2 to 0 mg/cm2 in T. latifolia and P. australis respectively.
A gradual increase in the total chlorophyll was observed from
0.075 mg/cm2 to 0.077 mg/cm2 and 0.026 mg/cm2 to 0.029 mg/cm2
in T. latifolia and P. australis respectively in 18 days at
35 C5000 lx combination. Similarly when plants were exposed to
35 C10 000 lx combinations the total chlorophyll increased from
0.073 mg/cm2 to 0.075 mg/cm2 in T. latifolia in 11 days and from
0.028 mg/cm2 to 0.029 mg/cm2 in P. australis in 12 days. The highest
increase in Chlorophyll was determined at 35 C15 000 lx which
121
was 0.064 gm/cm2 to 0.074 g/cm2 and 0.029 g/cm2 to 0.034 g/cm2
in T. latifolia and P. australis respectively. Whereas when plants
were provided with 35 C20 000 lx combination the chlorophyll
content decreased from 0.068 mg/cm2 to 0.025 mg/cm2 in T. latifo-
lia and from 0.028 mg/cm2 to 0 mg/cm2 in P. australis.
The total chlorophyll decreased from 0.066 mg/cm2 to
0.018 mg/cm2 in T. latifolia and from 0.027 mg/cm2 to 0 mg/cm2 in
P. australis when exposed to 40 C5000 lx. Similarly it decreased
from 0.068 mg/cm2 to 0.018 mg/cm2 and 0.027 mg/cm2 to
0 mg/cm2 in T. latifolia and P. australis respectively when plants
were exposed to 40 C10 000 lx. Likewise a decrease in the
chlorophyll was observed from 0.066 mg/cm2 to 0.017 mg/cm2
and from 0.026 mg/cm2 to 0 mg/cm2 in T. latifolia and P. aus-
tralis respectively when exposed to 40 C15 000 lx. Moreover a
decrease in the total chlorophyll was observed from 0.067 mg/cm2
to 0.015 mg/cm2 and 0.027 mg/cm2 to 0 mg/cm2 in T. latifolia
and P. australis correspondingly when exposed to 40 C20 000 lx
combinations (Fig. 8).
Thus it is evidenced that the combinations 25 C5000 lx;
25 C10 000 lx; 25 C15 000 lx are not optimum for both T. lati-
folia and P. australis, however the plants were able to survive and
did not deteriorate till the end of experimentation. Moreover light
intensity of 20 000 lx and temperature 40 C is proved to be critical
and harmful for both T. latifolia and P. australis. The chlorophyll at
such combinations reached to 0 in P. australis proving their lower
tolerance as compared to T. latifolia which did not ourished but
were able to survive till the end of the experiment.
122 F. Rehman et al. / Ecological Engineering 102 (2017) 112126

Fig. 10. Initial and nal fresh plant biomass of T. latifolia (A) and P. australis (B) at variable temperature and light intensity combinations under which plants survived and
ourished. The combinations which deteriorated the plants were excluded. The mean values of fresh plant biomass presented in the gure were taken before and after the
18 days experimentation. Different alphabetical letters indicate the signicant difference among nal fresh plant biomass.

These results are similar to the Nikolic et al. (2009) and Dar
et al. (2013), which indicated that the higher temperatures and light
intensities up to the certain extent during summer season caused
the increase in chlorophyll content of the P. Australia and T. angus-
tata and vice versa. It has been already proved that both light and
temperature act together in chlorophyll content of macrophytes
(Robledo and Freile-pelegrin, 2005) as combining the critical and
favorable temperature or light intensity did not assisted the plant
growth and DO release. Moreover photosynthesis is dependent on
chlorophyll content of the plant. Thus changes in chlorophyll con-
tent correspond to the rate of change or photosynthesis (Blackburn,
2007) and consequently the change in the release of oxygen from
the roots of macrophytes.
3.4.1. Correlation of DO with chlorophyll
The current study showed a positive linear correlation between
DO in the substratum of CWs and total chlorophyll in the leaves
of T. latifolia and P. australis, when plants were exposed to
30 C10 000 lx (Fig. 9a; Fig. 9A), 30 C15 000 lx (Fig. 9b; Fig. 9B),
35 C10 000 lx (Fig. 9c; Fig. 9C) and 35 C15 000 lx (Fig. 9d;
Fig. 9D) correspondingly. The increase in the amount of DO was
found associated with the increase in total chlorophyll content of
the leaves.
3.5. Effect of temperature and light intensity combinations on
fresh plant biomass of macrophytes
The results showed that the fresh plant biomass in T. latifo-
lia (Fig. 10A) did not increased or decreased at 25 C5000 lx;
25 C10 000 lx; 25 C15 000 lx; 30 C5000 lx and 35 C5000 lx.
However it decreased signicantly at 25 C20 000 lx;
30 C20000 lx; 35 C20 000 lx; 40 C5000 lx; 40 C10 000 lx;
40 C15 000 lx and 40 C20 000 lx. Moreover the fresh plant
biomass increased signicantly during the experiment when
exposed to 30 C10 000 lx (P < 0.001) and 30 C 15 000 lx
(P < 0.001) with no signicant difference between both combina-
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126 123

Fig. 11. Correlation between DO and COD removal at variable temperature and light intensity combinations when CWs with T. latifolia were exposed to (a) 30 C10 000 lx;
(b) 30 C15 000 lx; (c) 35 C10 000 lx; (d) 35 C15 000 lx and CWs with P. australis were exposed to (A) 30 C10 000 lx; (B) 30 C10 000 lx; (C) 35 C10 000 lx; (D)
35 C15 000 lx. Mean values of COD and DO are recorded on day to day basis. Initially lower level of DO increased as the COD level decreased and became stable with the
stable value of COD.

tions. The highest increase in fresh plant biomass was observed
in T. latifolia at 35 C10 000 lx (P < 0.001) and 35 C15 000 lx
(P < 0.001).
Similarly P. australis (Fig. 10B) decreased their fresh plant
biomass at 25 C5000 lx; 25 C10000 lx; 25 C15000 lx and
25 C-20000 lx. The combinations i.e. 40 C500C5000 lx;
40 C10000 lx; 40 C15000 lx and 40 C-20000 lx deteriorated
the plants completely. Conversely the fresh plant biomass was
signicantly increased at 30 C500C5000 lx (P < 0.001). A further
signicant increase was observed at 30 C10000 lx (P < 0.001);
30 C15000 lx (P < 0.001) and 35 C10000 lx (P < 0.001). However,
the highest increase in the fresh plant biomass was observed at
35 C15000 lx (P < 0.023).
Moreover the temperature 40 C and light intensity 20000 lx
caused withering and leaf fall in P. australis. The initial, day 10
and nal fresh plant biomass of the T. latifolia and P. asutralis are
mentioned in Table 5.
Thus it is clearly determined that the fresh plant biomass of
both plants was affected by the combinations of temperature and
light intensity. In T. latifolia the combinations 25 C500C5000 lx;
25 C10000 lx; 25 C15000 lx; 30 C5000 lx and 35 C5000 lx
were identied not favorable for the plant growth although the
plants were able to survive. Moreover the deterioration and sig-
nicant decrease in plant biomass in T. latifolia at combinations
with temperature 40 C and light intensity 20 000 lx signies these
factors as critical for the plant growth. There was no release of
DO from their roots and the plants were deteriorated during the
experiment. However a substantial increase in fresh plant biomass
at 30 C10 000 lx; 30 C15 000 lx and further signicant increase
at 35 C1000 lx and 35 C15 000 lx indicates these combinations
encouraging for the photosynthesis and plant growth in T. latifolia.
124 F. Rehman et al. / Ecological Engineering 102 (2017) 112126

Fig. 12. Correlation between DO and BOD removal at variable temperature and light intensity combinations when CWs with T. latifolia were exposed to (a) 30 C10 000 lx;
(b) 30 C15 000 lx; (c) 35 C10 000 lx; (d) 35 C15 000 lx and CWs with P. australis were exposed to (A) 30 C10 000 lx; (B) 30 C10 000 lx; (C) 35 C10 000 lx; (D)
35 C15 000 lx. Mean values of BOD and DO are recorded on day to day basis.

It was also evidenced that at highest temperature and light
intensity T. latifolia showed slight and gradual decrease in the
biomass however plants were not completely depreciated till the
end of the experiment showing higher tolerance of T. latifolia to
extreme temperatures and light intensities as compared to P. aus-
tralis which were completely destroyed till the end of 18 days
experiment.
The total plant biomass increased at medium light intensities.
The lower and intense light intensities prove to be unfavorable for
the plant growth and consequently for the plant biomass. How-
ever the response of different macrophytes species may vary for
certain environmental factor. The inhibitory effect of unfavorable
environmental conditions like soil redox potential on photosyn-
thesis, growth of the macrophytes and consequently the oxygen
release from the roots have also been studied by many scientists
and was observed that at unfavorable soil redox potential the pro-
cess of photosynthesis was slowed down and thus plant biomass
was also reduced, which affected the release of oxygen from the
roots of macrophytes (Pennington and Walters, 2006; Lissner et al.,
2003).
3.6. Effect of macrophyte exposure to temperature and light
intensity combinations on removal efciency of CWs and HRT
The COD and BOD percent removal efciencies of vegetated and
non-vegetated CWs are along with the HRT are summarized in
Table 6.
Although over 95% removal efciency was attained by all vege-
tated and non- vegetated CWs but the retention time was increased
in non-vegetated CWs. The results of the present study are similar
to the results of which showed that the time required for the treat-
ment of wastewater with P. australis was less than the time utilized
by the CWs without P. australis. That approves the important role
of the plants in purication process. The present study also iden-
tied that the vegetation in the wetland systems had a substantial
role in treatment processes but in the absence of plants, the gravel
substrate provided a noteworthy wastewater treatment (Hamilton
et al., 1993; Coleman et al., 2001) with greater retention time. When
plants in CWs were exposed to optimum temperature and light
intensity, the retention time was lowered, which indicated the role
of dissolved oxygen in organic matter removal from wastewater,
 F. Rehman et al. / Ecological Engineering 102 (2017) 112126 125

Table 5
Fresh plant biomass in grams per plant of T. latifolia and P. australis. Mean values were recorded before day 10 and after 18 day experiment. The day 10 fresh plant biomass
value is recorded from the plants in back up CWs to avoid any trauma and ongoing growth of the plants.

Temperature and light T. latifolia (g/plant) P. australis (g/plant)

intensity combination

Initial Day 10 Final Initial Day 10 Final

25 C5000 lx 118.7 119.2 121 194.3 185 178.3
25 C10 000 lx 120.1 121 120.1 188 189 174.4
25 C15 000 lx 121.2 122.2 129.3 182 171 162.4
25 C20 000 lx 117.4 117.8 83 197.5 94 67
30 C5000 lx 118.4 119.1 121.1 186.3 193 211.6
30 C10 000 lx 121.1 139.2 152.7 176.9 194 249.7
30 C15 000 lx 115.9 132.2 148.3 183.4 221 256.3
30 C20 000 lx 118.1 88 56 179.8 123 82.6
35 C5000 lx 119.5 119.7 122.1 175 181 201.5
35 C10 000 lx 120.2 134.8 156.3 189.3 249.4 281
35 C15 000 lx 116.9 142.3 161.7 177.6 223 276
35 C20 000 lx 118.7 84 67.8 209.4 123 67.2
40 C5000 lx 117.4 86 43.6 231.2 110 58.4
40 C10 000 lx 120.1 86 65.5 182.5 91 41
40 C15 000 lx 117.8 72 42.8 192.4 82 38.5
40 C20 000 lx 118.3 82 51.2 201.2 91 29.1

Table 6
Percent removal efciencies and HRT of vegetated and non-vegetated CWs at 16 different temperature and light intensity combinations.

Temperature and light Typha latifolia Phragmites australis Control

intensity combinations

COD% removal BOD% removal COD% removal BOD% removal COD% removal BOD% removal

25 C, 5000 lx 97.17% 97.42% 97.48% 97.90% 97.13% 97.63%
Days 14 15 17
25 C, 10 000 lx 97.07% 97.37% 96.72% 96.98% 97.38% 97.47%
Days 13 13 18
25 C, 15 000 lx 97.48% 97.80% 97.23% 97% 96.47% 97.10%
Days 14 13 17
25 C, 20 000 lx 97.09% 97.28% 97.05% 97.10% 96.97% 97.27%
Days 15 15 18
30 C, 5000 lx 97.22% 97.28% 96.98% 96.97% 97.60% 97.56%
Days 12 12 17
30 C, 10 000 lx 97.14% 97.58% 96.96% 96.59% 97.38% 97.65%
Days 10 10 16
30 C, 15 000 lx 97.54% 97.44% 97.48% 97.59% 96.92% 96.96%
Days 8 11 16
30C, 20 000 lx 97.26% 97.08% 97.49% 96.38% 97.61% 97.64%
Days 14 15 18
35 C, 5000 lx 97.67% 97.51% 97.59% 97.80% 96.72% 97.16%
Days 13 13 17
35 C, 10 000 lx 96.60% 97.26% 97.10% 97.37% 97.09% 97.52%
Days 12 9 16
35 C, 15 000 lx 96.71% 96.96% 97.20% 97.43% 96.68% 97.05%
Days 8 9 17
35 C, 20 000 lx 97.02% 97.06% 97.61% 97.56% 97.22% 97.53%
Days 15 15 17
40 C, 5000 lx 97.38% 96.75% 96.63% 96.82% 96.60% 97.16%
Days 15 14 17
40 C, 10 000 lx 96.87% 97.06% 97.61% 96.96% 97.12% 97.34%
Days 15 15 17
40 C, 15 000 lx 96.70% 97.05% 97.64% 96.98% 96.76% 96.73%
Days 15 15 17
40 C, 20 000 lx 96.98% 97.06% 97.88% 97.87% 96.97% 97.95%
Days 15 15 18

as the only source of oxygenation in the substratum was restricted intensity, the amount of oxygen release to the rhizosphere can be
to plants. enhanced to lower the retention time of the wetlands.
According to some scientists BOD removal in wetlands takes
place due to both physical and biological processes i.e. sedimen-
3.6.1. Correlation of DO, COD and BOD removal
tation and aerobic degradation by bacteria which are attached
A strong negative correlation between DO and COD (Fig. 7)
to the roots of plants (Tanner et al., 1995; Brix, 1997; Naylor
removal; DO and BOD was found (Fig. 8) in vegetated CWs
et al., 2003). The present study indicated that the macrophytes
with T. latifolia when exposed to 30 C10 000 lx (Fig. 11a;
by providing oxygen to the substratum of wetlands and habitat
Fig. 11A); 30 C15 000 lx (Fig. 11B); 35 C10 000 lx (Fig. 11C);
for micro-organisms involved in biodegradation played a vital role
35 C15 000 lx (Fig. 11d; Fig. 11D) correspondingly. Similarly a
in organic matter removal from wastewater. Furthermore by pro-
positive correlation was found between DO in the rhizosphere of P.
viding these macrophytes with optimum temperature and light
australis and COD; BOD removal when exposed to 30 C10 000 lx
126 F. Rehman et al. / Ecological Engineering 102 (2017) 112126
(Fig. 12A); 30 C15 000 lx (Fig. 12B); 35 C10 000 lx (Fig. 12C);
35 C15 000 lx (Fig. 12D) correspondingly. The generalized trend
was initially low DO concentration which gradually increased and
remained stable after specic time period, however the similar
trend was observed in COD and BOD removal which was higher
in the beginning, decreased gradually and remained stable at par-
ticular level (Lemounava and Komanava, 2013).
4. Conclusion
The current study established that the ideal temperature and
light intensity for T. latifolia to release the maximum DO from their
roots ranged between 30 C to 35 C and 15 000 lx respectively.
Whereas the best temperature for P. australis to release maxi-
mum oxygen from their roots was 35 C and optimal light intensity
ranged from 10 000 lx to 15 000 lx. Accordingly fresh plant biomass
and chlorophyll content of the plants were signicantly increased at
these optimal temperature and light intensity combinations. More-
over a strong positive linear correlation was found between DO and
chlorophyll content of the (both) plants at optimum temperature
and light intensity combinations. A strong negative linear corre-
lation between DO and hydraulic retention time (for both plants)
at optimum temperature and light intensity combination was evi-
dent. Thus it was concluded that the DO release from the roots of
the plants was associated with the process of photosynthesis and
adjusting the optimum temperature and light intensity favorable
for the photosynthesis assisted to increase the dissolved oxygen
released from the roots of the plants which consequently improved
the efciency of the constructed wetlands.
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