Growth and nutrient removal efficiency of duckweed (lemna minor) from synthetic and dumpsite leachate under artificial and natural conditions
Growth and nutrient removal efficiency of duckweed (lemna minor) from synthetic and dumpsite leachate under artificial and natural conditions
Jamshaid IqbalID 0 1
Atif JavedID 1
Muhammad Anwar Baig 1
0 Department of Environment and Energy Management, College of Business Management, Institute of Business Management (IoBM) , Karachi, Sindh , Pakistan , 2 Department of Environmental Sciences, University of Okara , Okara, Punjab , Pakistan , 3 Institute of Environmental Sciences and Engineering, School of Civil and Environmental Engineering, National University of Sciences and Technology , Islamabad , Pakistan
1 Editor: Bawadi Abdullah , Universiti Teknologi Petronas , MALAYSIA
Sustainable management of leachate produced from the dumpsite is one of the major concerns in developing countries Aquatic plants such as duckweed have the potential to remove pollutants from wastewater which can also be cost-effective and feasible options for leachate treatment. Therefore, the objective of our present study was to examine the growth and nutrient removal efficiency of duckweed (Lemna minor) on leachate. Three tests were performed each by growing lemna minor on synthetic leachate under controlled conditions and on dumpsite leachate under natural conditions. During each test, duckweed was grown in 300 ml plastic containers with a surface area of 25.8 cm2. About 60 mg of fresh mass of duckweed was grown on 250 ml leachate at an internal depth of 9.5 cm. Results revealed that, in comparison to synthetic leachate, duckweed removed Chemical Oxygen Demand (COD), nitrogen (N), and phosphorous (P) more efficiently from dumpsite leachate under natural climatic conditions. However, the amounts of N and P absorbed into duckweed body mass were about 16% and 35% respectively more at synthetic leachate under controlled conditions. Maximum growth rate of duckweed (7.03 g m-2 day-1) was also observed for synthetic leachate in comparison to the growth rate of 4.87 g m-2 day-1 at dumpsite leachate. Results of this study provide a useful interpretation of duckweed growth and nutrient removal dynamics from leachate under natural and laboratory conditions.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: Authors are grateful to Higher Education
Commission of Pakistan for providing financial
support during the execution of this research
study. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
The absence of standard landfill sites in developing countries has given rise to the formation of
open waste dumpsites which produce relatively large amounts of leachate [
]. Leachate is a
type of concentrated wastewater produced at open dumpsites by percolation of rainwater
through solid waste layers [
]. Composition of typical leachate is highly variable containing
large amounts of pollutants and nutrients such as organic matter, ammonia-nitrogen, heavy
metals, and chlorinated organic and inorganic salts [
]. Pollution caused by leachate is a
potential threat to the environment and human health . Unattended leachate is representing
a potential environmental risk to the surface as well as ground water quality [
]. There are
many studies on the effect of leachate pollution on human health, flora, fauna and ecosystems
At present in view of the implementation of stringent leachate discharge standards
worldwide, it has become a major research focus to explore the various methods of leachate
]. A wide range of physical, chemical, biological and combination of two or more
methods of leachate treatments are being practiced worldwide [
]. The potential method for
leachate treatment is determined by many factors such as, current waste disposal practices,
geographical location of landfill/dumpsites, local weather pattern, leachate composition and
economic concerns of leachate treatment .
Use of aquatic plants such as duckweed, water hyacinth and water lettuce etc. has been
recognized and getting more attention recently in wastewater treatment [
]. Aquatic plants also
offer an alternate technology of converting wastewater nutrients into potentially useful forms
in addition to the treatment [
]. Duckweed is amongst the promising aquatic plants having
enormous capacity to treat eutrophicated wastewaters. Wastewater treatment by duckweed is
owed to its ability to accumulate large amounts of nutrients and minerals into its body mass
and show high growth rates under worse environmental conditions [
Duckweed is a small floating macrophyte belonging to family Lemnaceae of
monocotyledonous plants. It has 37 species belonging to 4 genera: i) Lemna, ii) Spirodela, iii) Wolffia and,
iv) Wolffiella [
]. It is a simple plant having no stem or leaves. Major part of the plant
comprises a thallus called "frond" which is generally composed of chlorenchymatous cells having
air pockets called aerenchyma due to which duckweed floats on water. Duckweed may have no
root or one or more simple roots. Roots are photosynthetically active having chloroplast in it.
Roots of the duckweed plant help in nutrient uptake from water and stabilizes the plant [
Lemna minor, belonging to the genus Lemna is the most widely spread species of duckweed
which is extensively studied in wastewater treatment mainly due to its fast growth and high
nutrient removal efficiency [
]. Under favorable climatic conditions and nutrient balance in
growth media, Lemna minor can double its biomass within two days [
]. Cheng et.al reported
a growth rate of L. minor close to 29 g m-2 day-1 in high strength swine wastewater while the
total Kjeldahl Nitrogen (TKN) and Total Phosphorous (TP) absorbed by duckweed were 90%
and 88.6%, respectively [
Assimilation of nitrogen by duckweed fronds and roots appears to be the primary
mechanism of nitrogen fixation in plant. However, some portion of nitrogen is also absorbed into
duckweed biomass through associated N fixing cyanobacteria and algae grown in duckweed
]. Nitrate and ammonium are the main forms of available nitrogen for duckweed
however, the absorption of ammonium is 3 to 11 times greater than nitrates. Nitrogen is fixed
as protein in duckweed biomass [
]. Various studies report a variable amount of nitrogen
absorbed by duckweed. Zhang et al. reported that in wastewater with initial nitrogen
concentration of 12 mg N L-1 duckweed consumed nitrogen at the rate of 446 mg m-2 day-1 [
Another study reported the nitrogen absorption rate of 547?136 mg N m-2 d-1 by duckweed
Unlike other vascular plants, Lemna minor absorbs large amount of phosphorous into its
body mass [
]. However, compared to nitrogen, the phosphorous requirement of duckweed
is very small for optimum plant growth [
]. Phosphate (PO4-3) is the preferred form of
phosphorous uptake by the duckweed. Phosphorous makes up to 0.03 to 2.8% of a typical duckweed
dry mass whereas the nitrogen content is about 0.8 to 7.8% [
]. Duckweed can accumulate
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high amounts of phosphorous in its biomass due to which plant can maintain its growth in
waters with less amount of phosphorous. When duckweed dies, stored P in plant biomass is
readily available in the water [
]. Literature shows that Lemna minor has varying capacity of
P uptake under different environmental conditions. Duckweed shows optimum growth at
phosphorus concentration of 4 and 22 mg P L-1 of growth media [
]. Phosphorous uptake of
200 mg m-2 d-1 by Lemna minor is also reported when grown on swine wastewater [
Another study reported the phosphorous uptake of 13 to 58 mg P m-2 d-1 and revealed that
phosphorous uptake by duckweed was dependent on nitrogen concentration and depth of the
growth pond [
Protein contents of a typical duckweed may be as high as up to 45% of the total dry mass of
plants. Due to high protein content, the harvested duckweed is a potential food source for
human and animal feeds [
Based on its wastewater treatment potential, it is hypothesized that Lemna minor can also be
used as a cost effective and technically feasible option for leachate treatment. Therefore, present
study was designed with the objective to investigate the growth of duckweed and its efficiency to
remove COD and nutrients (N&P) from synthetic and dumpsite leachate. Attempts at searching
for literature reveals that currently a very small amount of research has been conducted on the
use of duckweed for leachate treatment. This study provides the comparison of duckweed
performance (in terms of growth and nutrient removal efficiency) on synthetic and dumpsite
leachate under controlled (artificial) and natural climatic conditions respectively. So far, no such
comparison is available in literature however very few isolated research studies have been
conducted using either natural or artificial duckweed-leachate systems. Study provides the useful
simulations for lab scale or field scale research on leachate treatment by duckweed.
Materials and methods
Dumpsite leachate used in this study was prepared by processing the mixed solid waste
collected from various residential, commercial and industrial dumpsites in Islamabad, Pakistan.
About 100 to 120 kg of well decomposed solid waste was collected from each dumpsite. Waste
was collected from pre-determined lowest points at depths of 0.5 m to 1.5 m [
wastes were mixed in plastic tank having an internal diameter of about 1.5 m and a height of
about 1.8 m. A sieve (pore size 1mm) was fixed at an internal height of 10 cm of the plastic
tank. Fig 1 shows the schematic setup used for leachate production.
Synthetic leachate with COD about 1527?2.42 mgL-1 (approximately equal to the COD of
dumpsite leachate) was prepared by adding the measured quantities of NaNO3, K2HPO3,
KHCO3, K2CO3, NaHCO3, MgCl2.6H2O, MgSO4.7H2O, CaCl2 and glucose powder in distilled
water. In view of the complex chemical composition of dumpsite leachate, it was difficult to
prepare synthetic leachate of exactly similar composition. However, after repeated
measurements and hit and trial analysis synthetic leachate with desired COD, and nutrient contents
was prepared. Table 1 represents the nitrogen and phosphorous contents and COD of
synthetic leachate. Because leachate composition shows high temporal variability [
an initial analysis of both types of leachate were made soon after the preparation and the
analyzed leachate was immediately used for duckweed growth.
A mixed culture of duckweed was collected from wastewater treatment pond located in
National University of Sciences and Technology (NUST), Islamabad, Pakistan. Lemna minor
plants were isolated from the mixed duckweed culture and used for this study after
acclimatization for about seven days.
Three separate tests (each comprising two parts) were performed during the month of
June-July by growing duckweed on dumpsite leachate (Part 01) under natural conditions and
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Fig 1. Schematic of leachate production setup.
on synthetic leachate (Part 02) under the similar artificial conditions. During each test,
duckweed was grown in 300 mL plastic containers with a surface area of 25.8 cm2. About 60 mg of
fresh mass of duckweed was grown on 250 mL leachate at an internal depth of 9.5 cm. During
each part of the three tests, nine (09) containers were used in triplicate including six duckweed
containers and three controls without duckweed.
For part 01 of each test, duckweed containers were placed within a meshed iron rack under
natural climatic conditions whereas; for part 02, containers were placed within the growth
chamber under similar controlled conditions. In the growth chamber, the required light
intensity was adjusted with the help of fluorescent lamps whereas, the required day lengths were
adjusted with the help of auto shut down system of fluorescent lights. Temperature
adjustments within the growth chamber were made with the help of a temperature gauge fitted with
the chamber. Throughout the experiments, the pH of the leachate was maintained at about 7?
0.6. using 1M solution of Hydrochloric Acid (HCl) and Sodium Hydroxide (NaOH).
Data related to ambient air temperature and day lengths as shown in Table 2 was retrieved
from the website of Pakistan Metrological Department, whereas the solar radiation data was
obtained from the web site of LEO Corporation, Pakistan.
Each test was performed for 10 days during which samples from the leachate and control
containers were analyzed for TKN, ammonium nitrogen (NH4+-N), TP, ortho phosphate
Ambient temperature (oC)
(kWh m-2 day-1)
4.3 ? 0.6
4.5 ? 0.5
4.4 ? 1.3
4.4 ? 0.8
13.4 ? 0.4
13.6 ? 0.4
14.1 ? 0.7
13.7 ? 0.5
(o-PO4-3-P) and COD by removing three containers at the start and end of the test. Duckweed
plants were oven dried at 70 0C until it had a constant weight. Dried mass of duckweed was
ground with the help of a mortar and pestle and then a plant extract was prepared for analysis
of TKN and TP contents. Furthermore, a chemical analysis was performed using the standard
methods of American Public Health Association [
]. The details of material used, and
experimental techniques adopted during study are given in Table 3.
All experiments during this study were conducted within the premises of Institute of
Environmental Sciences and Engineering (IESE), National University of Sciences and Technology,
Islamabad, Pakistan where I am pursuing my doctoral degree (33? 380 41@ N, 72? 590 22@ E).
Experimental site is owned by the IESE, NUST where no permits are required to conduct the
research work for IESE students. Furthermore, it is to note that no endangered or protected
species or locations were involved during this research study.
All treatments were performed in triplicate. Data collected on all parameters was analyzed
statistically using Fisher?s analysis of variance (ANOVA) techniques under completely
randomized design (CRD). Statistical analysis was performed using Statistix-8.1 and MS excel
Results and discussion
Table 1 depicts that the nutritive composition and COD level of synthetic and dumpsite
leachate has no significant difference. Therefore, for further data analysis and results interpretation,
initial composition of both types of leachates is assumed to be identical. Table 2 shows that
during each test there exists a very small difference in natural weather conditions. This
situation was also helpful for maintaining the artificial weather conditions in the growth chamber
where no significant variations of conditions was required throughout the experimental
Table 4 provides a comparison of duckweed growth rates at dumpsite and synthetic
leachate indicating that during each test, duckweed exhibited the better growth in synthetic
leachate than that of in dumpsite leachate. In synthetic leachate a maximum growth rate of
7.03 ? 1.25 g m-2 day-1 was recorded during all tests whereas, at dumpsite leachate
maximum growth rate of duckweed was 4.87 g m-2 day-1. Lemna minor has variable growth rates
under varying climatic conditions. Seasonal growth of duckweed ranges from 3 to 9.5 tons/
] whereas, the maximum yield of 17?25 tons/ac-year is also reported [
dumpsite leachate growth rates of Lemna minor ranging from 4.3 to 6.4 g m-2 day -1 have
also been reported [
]. Similar growth rates of duckweed (3.2 to 5.7 g m-2 day -1) were also
reported by another study conducted by growing duckweed on a dumpsite leachate under
varying electrical conductivities of leachate [
]. In dumpsite leachate under the natural
conditions, large amount of nutrients is removed by the other factors than absorption into
duckweed biomass which results in retarded growth of duckweed plants [
]. This might be
the reason of high growth rate of duckweed at synthetic leachate. Significant amounts of N
and P may be removed through ammonia volatilization, nitrification and denitrification
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Chemical Oxygen Demand (COD)
Total Kjeldahl Nitrogen (TKN)
Ammonium nitrogen (NH4+-N)
Total Phosphorous (TP)
Semi-Automatic Kjeldahl Distillation System
UV Visible Spectrophotometer &
UV Visible Spectrophotometer
UV Visible Spectrophotometer
Duckweed growth under controlled conditions
Velp ECO 25
PG-Motel T 60
PG-Motel T 60
Hanna HI 8520
Eutech pH 700
Adam AAA 160 LE
Adventure AR 3130
Chasewood, Environmental USA
and microbial assimilation in addition to the duckweed absorption in natural
leachateduckweed systems [
A comparison of COD and nutrients removal from synthetic and dumpsite leachate is
provided in Table 5 indicating that during each test, compared to synthetic leachate, duckweed
removed nutrients and COD more rapidly from dumpsite leachate under natural conditions.
Duckweed absorbs variable amounts of nitrogen and phosphorous under varying
conditions. Absorption of nitrogen by duckweed largely depends on the initial nitrogen
concentration in growth media, Chemical Oxygen Demand (COD), hydraulic retention time and
duckweed plant density [
]. Iqbal and Baig reported that during summer season Lemna
minor removed TKN and TP from dumpsite leachate at the rates of 40 to 310 mg m-2 day -1
and 30 to 200 mg m-2 day-1 respectively [
]. While TKN and TP removal of 152 to 175 mg
m-2 day -1 and 84 to 92 mg m-2 day -1 respectively by Lemna minor from dumpsite leachate
under natural conditions have also been reported [
]. The high rates of nutrients and COD
removal from dumpsite leachate are attributed to the processes such as ammonia
volatilization, algal and microbial assimilation, and nitrification/denitrification which are high
under natural duckweed-leachate systems [
]. Nitrification and denitrification processes
contributes for 50% of nitrogen removal from wastewater [
]. High rates of nitrification
and denitrification processes resulted by large population of respective bacteria may remove
large amounts of nitrogen from dumpsite leachate.
Growth rate (g m-2 day-1)
4.06 ? 1.18
6.84 ? 2.13
4.87 ? 1.62
7.03 ? 1.25
3.89 ? 0.78
6.77 ? 0.93
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Fig 2. Comparison of mass balance of total nitrogen and phosphorous removal and uptake by duckweed from
synthetic and dumpsite leachate.
It is evident from the comparison of mass balance that duckweed absorbed larger amounts
of nitrogen and phosphorous into its biomass from synthetic leachate as compared to the
absorption of these nutrients from dumpsite leachate under similar conditions (Fig 2). This is
consistent with the growth of duckweed which is also high at synthetic leachate.
This study provides the comparison of duckweed (Lemna minor) growth and its efficiency to
remove COD and N & P from synthetic and dumpsite leachate. Results reveal that compared
to synthetic leachate under artificial conditions, duckweed removes COD and nutrients more
efficiently from dumpsite leachate. However, the amount of nitrogen and phosphorous
absorbed into duckweed body mass was about 16% and 35% respectively more at synthetic
leachate. The high growth rate of duckweed was also observed at synthetic leachate. In
conclusion, many factors such as microbial activities, algal growth and natural decomposition also
contribute to nitrogen and phosphorous removal from leachate in addition to absorption by
duckweed under the natural duckweed-leachate system.
S1 File. Nitrogen mass balance.
S2 File. Phosphorous mass balance.
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Authors are grateful to Higher Education Commission of Pakistan for providing financial
support during the execution of this research study. We are also thankful to Water and
Wastewater Laboratory at Institute of Environmental Sciences and Engineering (IESE), National
University of Sciences and Technology (NUST), Islamabad, Pakistan for providing technical
support to conduct this research.
Conceptualization: Jamshaid Iqbal.
Data curation: Jamshaid Iqbal, Atif Javed.
Formal analysis: Jamshaid Iqbal.
Investigation: Jamshaid Iqbal.
Methodology: Jamshaid Iqbal.
Supervision: Muhammad Anwar Baig.
Writing ? original draft: Jamshaid Iqbal.
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