MANOHARBHAI PATEL INSTITUTE OF

ENGINEERING AND TECHNOLOGY

GONDIA

F O R W A R D I N G L E T T E R

Forwarded herewith to the Rashtrasant Tukdoji Maharaj Univesity, Nagpur,

and the dissertation

STUDIES ON TREATMENT OF WASTE WATER BY

PYTOREMEDATION PROCESS

Submitted by- Sandeep P. Ajmire , in partial fulfillment of the award of

the degree of Master of Technology in Environmental Engineering.

Prof. A. L. Nashine Prof. Dr.S.S. Rathor

Head of department Principal

Dept. of Civil Engg. MIET Gondia

MIET, Gondia

1

MANOHARBHAI PATEL INSTITUTE OF

ENGINEERING AND TECHNOLOGY

GONDIA

C E R T I F I C A T E This is to certify that dissertation entitled

STUDIES ON TREATMENT OF WASTE WATER BY

PYTOREMEDATION PROCESS

Submitted by Sandip P.Ajmire , in practical fulfillment of the

requirement for the award of Degree of Master of Technology in Environmental

Engineering to The Rashtrasant Tukdoji Maharaj University, Nagpur , is

bonafide research work carried out under my supervision and guidance. The

work embodied in this dissertation has not submitted previously for the award of

any degree or diploma.

2

Prof . A.M. Deshpande Prof.A.L. Nashine

Supervisor Head Of Department

Dept. Of Civil Engineering Dept. Of Civil Engineering

MIET GONDIA MIET GONDIA

A C K N O W L E D G E M E N T

I express my profound gratitude towards Prof. A.M. Deshpande

,Lecturer , Department of Civil Engineering. MIET Gondia, for this able

guidance.

I am extremely Grateful to Hon President Mr. Bupesh Kulmethe &

CEO A.V. Dhoke, Municipal Council Gadchiroli . Mr .M.G. Nisal , Lab

Asst . Environmental Engineering Lab MITE ,Gondia , without whose help the

project might have been completed. Mr. S.P. Waghmare, Executive Engineer

Jeewan Pradhikarn Gadchiroli & his technical and non technical staff, without

whose help the project might have been completed.

I express heartfelt thankful to Prof. Dr. S.S. Rathod , Principal & Prof

A.L.Nashine, H.O.D., Civil Engineering & Prof. P.E.Mishra Coordinate, PG

Deptt. Of Environmentel Engg.,MIET, Gondia, for providing necessary

facilities in the completion of this work and for his constant encouragement.

Sandeep P. Ajmire

3

C O N T E N T S

INTRODUCTION

1.1. General 1-11

1.2 . Pollution Problem

1.3 . Standards of Disposal

1.4. Treatment methodology

1.5. Objective and scope of study

LITERATURE REVIEW

2.1. General

2.2. Characteristics of domestic waste water

2.3. Treatment Processes

2.4. Process selection criteria for treatment of various domestic

waste water

2.5. Application of Phytoremedation to domestic waste water

PHYTOREMEDATION

3.1. History & back round

4

3.2. Definition & types of Phytoremedation

3.3.Introduction of Phytoremedation by Lemna

3.3.1

3.3.2 Factor influencing startup process of

phytoremadation by lemna.

3.3.1 Start up of Phytoremedation by Lemna

3.3.2 Factor influencing startup process of

phytoremadation by lemna.

3.3.3

3.3.4

3.3.7 Scope of phytoremadation by Lemna.

3.3.8 Design consideration for phytoremadation 3.3.3

3.3.4 DWT system design principles

3.3.5 Advantages of phytoremadation.

3.3.6 Disadvantage of phytoremadation.

3.3.7 Scope of phytoremadation

3.3.8 Design consideration for phytoremadation

3.3.9

3.4. Scope of phytoremadation

3.5

PLANTS AND METHOD

4.1 Cultures

4.2 Tested chemicals

4.3 Lemna bioassay

4.5

4.6

4.7

5

4.8

4.9

4.9.1

4.9.2

4.9.3

4.9.4

4.9.5

4.9.6

4.9.7

4.10

OBSERVATIONS ,RESULTS,AND DISCUSSION

5.1 OBSERVATIONS

5.2 RESULTS

5.3 DISCUSSION

5.4

5.5

5.6

5.7

Reference:

PHOTOGRAPHS

6

I N T R O D U C T I O N

1.1.General

The population of glob is increasing, the problem of municipal & industrial waste

tedious day by day. The legacy of rapid urbanization, industrialization, fertilizer &

pesticide use has resulted in major pollution problems in both terrestrial and aquatic

environments. In developing countries is major problem to treat the polluted water

from above sources. Chemical & mechanical menace are used for this purpose is

expensive. In response, conventional, remediation systems based on high physical

and chemical engineering approaches have been developed and applied to avert or

restore polluted sites. Much as these conventional remediation systems are efficient,

they are sparsely adopted because of some economical and technical limitations.

Generally, the cost of establishment and running deter their use and meeting the

demand particularly in countries with week economy. Logical this high cost

technology can neither be applied justifiably where

1. The discharge is abruptly high for short time but the entire average load is

relatively small.

2. The discharge is very low but long term (entire load is medium).

3. The discharge is continuously decreasing over a long duration.

Thus conventional remediation approaches are best for circumstances of high

pollutants discharge like in industrial mining and domestic waste water. Recently , it

is evident that durability restoration and long term contamination control in

conventional remediation is questionable because in the long run the pollution

problem is only is suspended or transferring from one site to another.

7

The efficiency of duckweed (Lemna gibba L.) as an alternative cost effective

natural biological tool in wastewater treatment in general and eliminating

concentrations of both nutrients and soluble salts was examined in an outdoor aquatic

systems. Duckweed plants were inoculated into primary treated sewage water systems

(from the collector tank) for aquatic treatment over eight day’s retention time period

under local outdoor natural conditions. Samples were taken below duckweed cover

after every two days to assess the plant’s efficiency in purifying sewage water from

different pollutants and to examine its effect on both phytoplankton and total and

fecal coli form bacteria.

The Lemnaceae family consists of four genera (Lemna, Spirodela, Wolffia &

Wolffiella) and 37 species have been identified so far. Compared to most other plants,

duckweed has low fiber content (about 5%), since it does not require structural

tissue to support leaves and stems. Of these, applications of Lemna gibba L

(duckweed) in wastewater treatment was found to be very effective in the removal

of nutrients, soluble salts, organic matter, heavy metals and in eliminating suspended

solids, algal abundance and total and fecal coli form densities. Duckweed is a floating

aquatic macrophyte belonging to the botanical family Lemnaceae, which can be

found world-wide on the surface of nutrient rich fresh and brackish waters. Outdoor

experiments to evaluate the performance of the duckweed as a purifier of domestic

wastewater in shallow mini-ponds (20 & 30 cm deep) showed that quality of resultant

secondary effluents met irrigation reuse criteria. Wastewater ammonia was converted

into a protein rich biomass, which could be used for animal feed or as soil fertilizer.

The economic benefit of the biomass by-product reduced wastewater expenditures to

approx. US$ 0.05 per treated m3 of wastewater, which was in the range of

conventional treatment in oxidation ponds.

The present study was concerned with decreasing pollution of municipal waste

waster up to degree Standards of Disposal as per National pollution control board.

1.2 . Pollution Problem

Municipal wastewater is producing in a huge quantity in most the cities of the

country that contain a diverse range of pollutants including ,the quality of municipal

wastewater of stagnant/ slow velocity may create problem of high epidemics of

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malaria & other water born diseases. Heavy Metals ,Oil and Grease ,Phenols,

Sulphide, Sulphate ,Nitrate ,Phospate, Dissolved Solids, Suspended Solids, COD,

BOD, which its disposal and treatment has become a challenge for the municipalities.

Many of the municipalities in growing cities neither have proper disposal system nor

have any treatment facility due to higher cost and in such a situation municipal

wastewater is discharge in to aquatic bodies like river, ponds and lakes, where it is

posing a serious threat to the water quality and become a big environmental problem.

1.3 . Standards of Disposal

In order to protect the environmental Govt. of India established pollution

control boards. Tolerance limit for the industrial effluent as per the environmental

protection act 1986 of Govt. of India shown in table 1.1 governs the check for the

pollution effect. In addition to these standards Maharashtra Pollution Control Board

has introduced tolerance limit for the dissolved oxygen as 5 mg/l, the minimum

should be maintained in the river course, 15 m from the discharge point of the effluent

in the river.

1.4. Treatment methodology

Primary treated sewage water were transferred to the laboratory from the

tertiary sewage water treatment plant after the preliminary sieving step to get rid of

large suspended solids. The transferred water was immediately collected into two

opaque tanks (as replicates) to prevent light entering except at the top (Parr et al.,

2002), each tank with dimensions of 50 cm long, 35 cm wide and 25 cm deep and was

filled with 25 L primary treated sewage water. Duckweed (Lemna gibba L.) plants

ere collected from Ganabiet-Tersa drain. The stock were cleaned by tap water then

washed by distilled water inocula of Lemna plants were transferred to the water

systems for aquatic treatment. The experiment was kept under outdoor local

environmental conditions for eight days retention time.

Water sampling. Subsurface (under duckweed mat) water samples for physico-

chemical, biological and bacteriological parameters were collected in polyethylene

bottles from all sides of tank and then mixed. This procedure carried out every 2 days.

9

Samples volume taken every two days for each of phytoplankton count and

chlorophyll a determination was 100 ml.

Parameters measured. Physico-chemical analyses (Table ) were carried out

according to standard methods for e examination of water and wastewater (APHA,

1992). Field parameters (pH, conductivity & dissolved oxygen) were measured in situ

using the multi-probe system (model Hydralab-Surveyor) and rechecked in

laboratory using bench-top equipment to ensure data accuracy for biological

parameters including total coli form count and fecal coliform. count, phytoplankton

identification and counting and chlorophyll a determination.

Determination of duckweed growth rate. This was determined for fresh and

dry weights. Samples of 20 cm2 area of Lemna plants were harvested periodically at

the designated time periods (every 2 days) and filtered using filter papers then fresh

weights were determined. These samples were then dried at 60oC for 48 h to a

constant weight and then dry weights were calculated.

Duckweed organic nitrogen content was estimated at the beginning of the

experiment and after 8 days retention time, then the obtained values were multiplied

by 6.25 to obtain protein content values.

1.5. Objective and scope of study

Physical, chemical, and biological technologies have been developed to treat

waste water and restore environmental quality; However their costs are high and most

of them are difficult to use under field conditions, hence in such a condition there is

an urgent need to study natural, simple, and cost-effective techniques for control

pollution from municipal & industrial effluents and treating such wastewater, such as

phytoremedation .

Viewing this fact Phytoremediation was assumed to be very useful, as it is an

innovative, eco-friendly and efficient technology in which natural properties of plant

is used in engineered system to remediate hazardous wastes through physical,

chemical, and biological processes from wastewater and sewage.

Phytoremedation is the utilization of plants accumulation capabilities to remove

contamination from water, soil and air, the capacity of aquatic plants to remove

pollutants from water is well documented.

The recent application of phytoremediation technology by duckweed in

wastewater treatment and management is quite interesting and revealing.

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Phytoremediation systems by duckweed are one of the options that have been widely

applied for combined handling of wastewater with the nutrients used for poultry and

aqua-cultural projects.

Lemna minor L. known as common duckweed is a small, free floating aquatic

plant fast growing, adapt easily to various aquatic conditions and play an important

role in extraction and accumulation of pollutants from waters [8]. In particular,

species of Lemna are reported to accumulate toxic metals and therefore are being used

as experimental model systems to investigate heavy metal induced responses,

Bioavaibility and bioaccumulation of various heavy metals in aquatic and wetland

ecosystems is gaining tremendous significance globally.

This study aimed to assessing the efficiency of duckweed (Lemna minor) in

phytoremediate the pollutants of wastewater. This natural accumulation is related with

the resistance which represents response of plants to metal stress conditions.

Duckweed commonly refers to a group of floating, flowering plants of the family

Lemnaceae. The different species (Lemna, Spirodela, Wolffia and Wolfiella) are

worldwide distributed in freshwater and wetlands, ponds and some effluents are the

most common sites to find duckweed. The plants are fast growing and adapt easily to

various aquatic conditions. They are able to grow across a wide range of pH, from pH

3.5 to10.5 but survive best between pH 4.5 to 8.3. The plants are found in temperate

climates and serve as an important food source for various water birds and fish.

Each plant species has different resistance and tolerance levels to different

contaminants. Therefore, several studies have been performed to elucidate heavy

metal toxicity to plants.

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TABLE 1.1

STANDRADS FOR WASTE WATER DISPOSAL

Sr.No. Parameters Standards

Inland

water

surface

Public

sewers

Land for

irrigation

Marine & costal

area

1 Colour &

odour

All efforts should be made to remove it as fact as possible

2 SS(mg/l) 100 500 200 i)100 for process

w.w.

ii) 10% above for

cooling water

effect.

3 pH 5.5 to 9.0

4 Temperature 40 45 -- 45 At discharge

5 Oil & grease

(mg/l)

10 20 10 20

6 Total

Nitrogen

100 -- -- 100

7 BOD 30 350 100 100

8 COD 250 -- -- 250

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2.1. General

The literature of Phytoremediation by lemna was collected from the studies

previously done by various persons. Their finding and suggestions are listed hear.

Various treatment methods are also discussed for the treatment of municipal waste

water with comparison of aerobic and anaerobic treatments. An application of

phytoremadation for waste water done by different persons and their findings are also

mentioned.

2.2. Characteristics of domestic waste water

Characteristic of waste water depend upon the raw material, process and

product made.

Oron et al. have study the waste water from ponds

Parameter Mean concentration in waste

water

Elimination

capacity %

Remark

Influent Effluent %

COD 500 320 30-40 Moderate

BOD 50 30 60 Good

Total N 40 20 50 Good

NH3 17 2 80-90 Excellent

Total P 6 3 50 Good

From the treatment point of view removal the parameters

Koner and Vermaat also established that Lemna gibba and microorganism

coexist with it reduced 75% of phosphate and plants used 52% for growth process and

this agreed with study of. Nayyef M. Azeez and Amal A. Sabbar, 2012. Efficiency of

Duckweed (Lemna minor L.) in Phytotreatment of Wastewater Pollutants from

13

Basrah Oil Refinery. Journal of Applied Phytotechnology in Environmental

Sanitation.

Korner et al. mentioned that duckweed significantly enhanced COD removal in

shallow batch systems reported that COD removal was in the range of 70% to 80% in

the discharged duckweed treatment system at Halisahar.

Lead (Pb),Copper (Cu),Cadimum (Cd) and Zinc (Zn) reached their minimum

concentrations of 0.2,0.02,0.02and 12 mg.L-1, respectively after 30 days, with a

reduction percentage of 98.7%, 99.8%, 99.6% and 72%, respectively, that was the

highest rates of reduction compared with other pollutants ,and this due to a plant's

ability to absorb metals and accumulated in their tissues.

Referred to the aquatic plants have the ability to accumulate essential metals

for their growth and development and these metals include iron, manganese, zinc and

copper.

Khellaf and Zerdaoui have proven through a laboratory experiment the

capacity of Lemna minor to tolerant high concentrations of copper, cadmium, nickel,

zinc, and the results of this study agreed with the results of Other studies in terms of

the capacity of aquatic plants on the accumulation of heavy metals and used it as

phytoremedator and monitors of heavy metals pollution .

Zimmo et al. found that BOD removal efficiency was higher in duckweed based

ponds than in algae based ponds.

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2.3. Treatment Processes

The different processing waste water various authors have suggested the methods of

treatment. The methods of treatments can be broadly classified as follows

A) Conventional methods of treatments

i) Biological methods

ii) Physiochemical method

iii) Land application method

B) Reuse of wastewater or by product recovery

C) Prevention of waste and waste strength reduction.

D) Specific approach.

2.4. Process selection criteria for treatment of various domestic waste

water.

Over the years, biological treatment has established as a cost-effective solution in

a wide variety of domestic wastewater management problems. It is therefore, desirable to

consider whether the waste is amenable to biodegradation or can be rendered

biodegradable. Once the biodegradability of the waste established. The most appropriate

method of biological treatment can be selected. The available bio treatment alternative

differ from one another in many respect such as nature of electron acceptor (aerobic,

anoxic, or anaerobic), biomass state (suspended or fixed growth), hydraulic regime (plug

flow or completely mixed), and others. Selection of process should, however, be based

primarily on the waste water characteristics and the treatment gols (W.W.Eckenfedr et.al

1989).

2.4.1 Factor affecting process selection.

The factors affecting process selection for natural treatment are the raw

wastewater characteristics and the treatment objective. Additional factors such as climatic

conditions, plant location, land availability, etc. also affect processes selection.

Wastewater Characterization: A classification of the organic present in the

domestic waste water into various fractions based on amenability to biological treatment.

The organics are relatively more easily removed in any biological processes they are

enmeshed in the biomass and either degrader or physically separate from the liquid. The

15

soluble organics are generally more difficult to remove since portion of these compounds

are not readily available to the biomass. Those soluble organic which are sorbed into

biomass are also removed with relative ease although part of such organics may degrade

rather slowly. Of the non soluble organic organic through the activity of extra cellular

enzymes, while a non degradable portion will be left in the effluent. Other waste water

characteristics of concern process selection are the organics concentration, the presence

of nutrients, toxicants or inhibitory compounds.

Treatment Objectives :-

Treatment Objectives also play an important role in process selection. The primary

treatment objective in biological system is removal of biodegradable organic to levels

specified by regulatory agencies. Different treatment process can be tailored to achieve

the desire level of organic removal, toxicity reduction and non- degradable organic

removal.

2.4.2 AVAILABLE BIOLOGICAL TREATMENT PROCESS :-

The essence of biological treatment is the utilization of organic pollutants by

microorganisms for growth and maintenance. This can be represented by the following

simplified equation.

Organics+Nutrients+Electron Acceptor = New Biomass +End Product +Energy

A schematic illustration of the most common biological treatment processes currently

available is presented in fig. 2.2 All biological treatment process can be generally

categorized as aerobic or anaerobic. In the former, molecular oxygen systems, oxidized

nitrogen serves as electron acceptor and is reduced to nitrogen gas.

Both aerobic and anaerobic processes can further be classified as fixed growth systems.

The most common aerobic fixed growth systems are the trickling filters and the rotating

biological contactors (RBC). The aerobic dispersed growth systems are the aerated

lagoons and activated sludge processes. The latter may assume different forms in terms of

hydraulic configuration such as plug flow, completely mixed etc. in special cases, pure

oxygen or nitrification / denitrification systems are used.

16

The anaerobic treatment can also be divided into fixed and dispersed growth processes as

shown in fig. 2.2. The dispersed growth system is also known as anaerobic contact

process and is similar to activated sludge except it does not use oxygen. The fixed

growth anaerobic system include fluidized beds and packed beds. A hybrid of fixed and

dispersed growth system is the up flow anaerobic sludge blanket (UASB) process.

Fig represents the major types of biological treatment processes that are currently

available. However wastewater characterization and establishment of treatment

objectives are necessary before screening and selection of the process. Some of the

criteria and rationale behind this procedure are discussed below.

2.4.3 AEROBIC VERSES ANAEROBIC TREATMENT :-

A general comparison of aerobic and anaerobic treatment process is presented in table

1.2. In the aerobic process, where oxygen is the electron accepter, the growth process is

more efficient. It therefore, results in higher sludge yields and energy requirements, but is

less likely to produces odours.

The anaerobic processes are more sensitive to environmental condition (pH, temperature

toxic shocks) and require longer start up time. One major limitation of the anaerobic

process is that it cannot economically achieve levels, such as en effluent BOD of 20mg/L

or 95% BOD removal, as often required by regulatory agencies it can be cost effective,

however, if employed as pretreatment before aerobic polishing of high strength industrial

wastewater.

2.4.4 DISPERSED GROWTH VERSUS FIXED –BED REACTORS:

It is convenient to divide biological, reactors into dispersed growth and fixed bed

reactors. Biodegradation is carried but by biomass that is suspended in the liquid phase of

the reactor. In the fixed bed reactor, the biomass is attached to a fixed within the reactor.

Compared to the dispersed growth to a fixed within the reactor. Compared to the

dispersed growth reactors, the primary merit associated with the fixed bed reactors stem

from their simplicity and ease of operations, thus making them ideal for remote and

small industrial streams. Furthermore, because of the relatively high concentration of the

biomass attached to the surface of the fixed media these reactors can handle higher loads

17

per unit volume of reactors. Therefore, they are a better choice whenever land is limited.

sludge of relatively constant nature that can readily be removed by sedimentation. This is

particularly important whenever sludge settling problems are expected in an alternative

suspended growth systems less affect fixed bed reactors.

The major disadvantages of the fixed be reactors compared to the dispersed growth

systems are their lesser flexibility in operation, difficulty to achieve very high removal

efficiencies, and greater sensitivity to cold weather conditions. Another important

drawback of fixed bed system is that they are less understood, thus modeling and process

design procedures are not as rigours and advanced as for the dispersed growth systems.

This drawback has two important implications. First, in many cases the fixed bed reactors

are improperly designed; which leads to either over or under design. Second, it is more

difficult to estimate prototype performance based on bench scale experiments. This kind

of draw back is of particular importance in cases where the nature of the wastewater is

unknown.

Since the achievement of high removal efficiencies in fixed bed systems is economically

prohibitive these systems are often utilized as a roughing stage preceding is dispersed

growth polishing stage.

2.4.5 HIGH RATE ANAEROBIC TREATMENT

All high rate anaerobic treatment processes are based on the achievement of a

high retention of viable anaerobic sludge, combined with a good contact between

incoming wastewater with the sludge. Although these conditions are not always

sufficiently met in the available high rate systems, the importance of high rate systems

for practice is considerable because of the following reasons.

Very high organic loading rates can be applied.

Consequently small reactor volumes suffice.

Unless designed at their maximum loading potentials the stability of high

rate systems to sub optimal conditions (lower temperature, shock loads,

presence of inhibitory compounds ) is high.

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They make anaerobic treatment economically feasible at low ambient

temperature and for very low strength wastes as well.

2.5. Application of Phytoremedation to domestic waste water

The ability of duckweed to sequester nitrogen and phosphorus, and in so doing

“cleanse” dirty water, has been widely discussed in the literature for nearly 30 years

(Culley and Epps, 1973; Hillman and Culley, 1978; Oran et al., 1986; Landolt and

Kandeler, 1987; Leng, 1999). Systems utilising various species of duckweed, either alone

, or in combination with other plants, have been used to treat primary and secondary

effluent in the U.S.A. (Zirschky and Reed, 1988), the Middle East (Oran et al., 1985) and

the Indian subcontinent (Skillicorn et al., 1993; van der Steen et al., 1998).

Notwithstanding this reputation, some species and isolates are apparently quite sensitive

to high levels of nitrogen and/or phosphorous (Bergman et al., 2000), and effluent with a

high biological oxygen demand (BOD), such as abattoir waste, may kill the plants.

19

Although duckweed has a reputation for absorbing large amounts of dissolved nitrogen,

the degree of absorption appears to vary with concentration of nitrogen, time, species,

and (at least in temperate zones) the season. There is also strong evidence that there is a

symbiotic, or at least a synergistic relationship between duckweed and bacteria, both in

the fixation of nitrogen (Duong and Tiedje, 1985), and the removal of Chemical Oxygen

Demand (COD) (Korner et al., 1998) from water.

Differences in methodology, scale, and the parameters, both recorded and

measured, make direct comparisons between the many trials in published literature

difficult. However most research indicates that duckweed removes 40 to 60% of nitrogen

in solution over a 12 to 24 day period. Volatilization may account for a similar loss of

nitrogen (Vermaat and Haniff, 1998), although recent work completed in Israel (Van der

Steen et al., 1998), has suggested that direct duckweed absorption may account for less

than 20% of nitrogen loss, and volatilization/ denitrification may account for over 70%

In a similar fashion, lemnacae are generally able to absorb 30 to 50% of dissolved

phosphorous, although one researcher (Alaerts et al., 1996) has claimed over 90%

removal in a working, full scale system.

Phosphorous uptake (as measured by tissue phosphorous) and crude protein,

increased linearly with increases in nutrient concentration, up to approximately 1.5 g P/l,

and increased in absolute terms, up to 2.1 g P/l (Sutton and Ornes, 1975). This was

recorded in conjunction with a proportional rise in nitrogen concentration, thus the

association between nitrogen and phosphorous concentrations was unclear. COD is a

measure that quantifies water quality as determined by dissolved oxygen. All research in

the use of duckweed for improving effluent quality has determined significant but

variable decreases in COD (Alaerts et al., 1996; Karpiscak et al., 1996; Bonomo et al.,

1997; Vermaatand Haniff, 1998; van der Steen et al., 1999). However, a substantial

decrease in COD would be expected in open ponds without the presence of duckweed

(Al-Nozaily et al., 2000), so this improvement may not be attributable to the actions of

duckweed. Simplistically, the duckweed’s environment is somewhat two-dimensional. In

practice, this means that once the surface of a body of water is completely covered, the

plant has limited further opportunities to grow. Thus, insituations where there are high

20

nutrient levels, the clearance of dissolved nutrients is likely to be limited by harvesting

rate.

The work of Whitehead et al. (1987) confirms that at high average nutrient levels

(short retention time), nitrogen and phosphorous removal is enhanced with increased

cropping rate, whereas low nutrient concentrations favour low cropping rates. This latter

state indicates that growth is limited by nutrient availability. Degradation of bacterial

pathogens is a complex process and a comprehensive discussion is beyond the scope of

the current paper. However, two groups conducting specific investigations into this

issue (Karpiscak et al., 1996; van der Steen et al., 1999) found that faecal coliforms

decreased by 50 to 90% and that Giardia and Cryptosporidium fell by over 80% in

eutrophic waters in which duckweed was grown.

21

3.1. History & back round

In industrial areas, especially near steelworks, working mines and closed mine,

the environment is polluted by toxic heavy metals. High concentrations of these elements

are also found along roads and motorways. In water environments these elements

accumulate in the organs of macro phytes, fatty tissues of fish species, and bottom

sediments (Wilson and Bell, 1996; Karczewska, 2002). Duckweed (Lemna minor L.) is

an aquatic plant living in many types of water ecosystems, including lakes, streams and

ponds. Because it floats on the water surface, it is exposed to both water and air

contaminants (Mohan and Hosetti, 1999). In the past it was thought that duckweed is

highly tolerant to toxic substances. Currently there are many suggestions that L. minor is

sensitive to xenobiotic substances. To explain this contradiction it has been suggested

that duckweed is highly adaptive to environmental toxicity (Gabrielson et al., 1990;

Mohan and Hosetti, 1999). Lemna minor can be used in phytotoxicity tests of

contaminants, including heavy metals, phenolics and herbicides (Vujevic et al., 2000).

Tests of heavy metal toxicity consist in measurements of growth parameters and

physiological and biochemical indicators, including changes in carbohydrate, protein and

chlorophyll content (Mohan and Hosetti, 1999). Experts from the U.S. Environmental

Protection Agency (EPA) and the Organization for Economic Cooperation and

Development (OECD) have classified this plant as a bioindicator (Kiss et al.,

2003).Symptoms of heavy metal toxicity are chlorosis, necrosis and root damage, as well

as changes in biochemicals including antioxidant enzymes. The sensitivity of L. minor

has been tested in terms of some metabolic indicators, in sewage ponds (Mohan and

Hosetti, 1999) and under laboratory conditions (Garnczarska and Ratajczak,2000a,b;

Wang et al., 2002). Since the data are not conclusive, duckweed’s potential as a

bioindicator for aquatic systems needs further investigation.

Duckweed commonly refers to a group of floating, flowering plants of the family

Lemnaceae. The different species (Lemna, Spirodela, Wolffia and Wolfiella) are

22

worldwide distributed in freshwater and wetlands, ponds and some effluents are the most

common sites to find duckweed. The plants are fast growing and adapt easily to various

aquatic conditions. They are able to grow across a wide range of pH, from pH 3.5 to10.5

but survive best between pH 4.5 to 8.3 (Environnement Canada, 1999; Cayuela et al.,

2007). The plants are found in temperate climates and serve as an important food source

for various water birds and fish (Drost et al., 2007). Some studies indicate that duckweed

plants are sensitive to toxicity. Other studies however, report that duckweed plants are

tolerant to environmental toxicity (Wang, 1990). To assess the tolerance of the species L.

gibba to heavy metals, plants were exposed to concentrations of copper and nickel

higher than those used in medium cultures. Toxic effect of pollutant on duckweed is

generally evaluated by phytotoxicity tests based on growth inhibition (Geoffroy et al.,

2004). Copper and nickel were chosen as the metals for this study for a number of

reasons. Their presence above trace levels in the environment is an indicator of industrial

pollution. On the other hand, they are essential micronutrients for plants; copper is a

structural and catalytic component of many proteins and enzymes involved in metabolic

pathways (Teisseire & Vernet, 2000) and nickel has an important role in the urease and

hydrogenase metabolism (Harish et al., 2008). However, when the concentration reaches

a threshold value, these essential metals become first inhibitory and afterwards toxic.

Copper is responsible for many alterations of the plant cell (respiration, photosynthesis,

pigment synthesis and enzyme activity) (Teisseire & Vernet, 2000; Kanoun-Boulé et al.,

2009). Nickel inhibits germination, chlorophyll production and proteins (Zhou et al.,

2009) in plants; several animal experimental studies have shown an increased cancer

incidence associated with chronic exposure to nickel.

3.2. Definition & types of Phytoremedation

What is phytoremadation ?

23

Phytoremediation is the use of living green plants

for in situ risk reduction and/or removal of contaminants

from contaminated soil, water, sediments, and air.

Specially selected or engineered plants are used in the

process. Risk reduction can be through a process of

removal, degradation of, or containment of a contaminant

or a combination of any of these factors. Phytoremediation is an energy efficient,

aesthically pleasing method of remediating sites with low to moderate levels of

contamination and it can be used in conjunction with other more traditional remedial

methods as a finishing step to the remedial process. One of the main advantages of

phytoremediation is that of its relatively low cost compared to other remedial methods

such as excavation. The cost of phytoremediation has been estimated as $25 - $100 per

ton of soil, and $0.60 - $6.00 per 1000 gallons of polluted water with remediation of

organics being cheaoer than remediation of metals. In many cases phytoremediation has

been found to be less than half the price of alternative methods. Phytoremediation also

offers a permanent in situ remediation rather than simply trans locating the problem.

However phytoremediation is not without its faults, it is a process which is dependent on

the depth of the roots and the tolerance of the plant to the contaminant. Exposure of

animals to plants which act as hyperaccumulators can also be a concern to

environmentalists as herbivorous animals may accumulate contaminates particles in their

tissues which could in turn affect a whole food web. 

How Does It Work?

24

Phytoremediation is actually a genneric term for several ways in which plants can

be used to clean up contaminated soils and water. Plants may break down or degrade

organic pollutants, or remove and stabilize metal contaminants. This may be done

through one of or a combination of the methods described in the next chapter. The

methods used to phytoremediate metal contaminants are slightly different to those used to

remediate sites polluted with organic contaminants.

Metal Organic

Phytoextraction Phytodegradation

Rhizofiltration Rhizodegradation

Phytostabilisation Phytovolatilisation

Methods of Phytoremediation

Phytoremediation of metal contaminated sites

Phytoextraction (Phytoaccumulation)

25

Phytoextraction is the name given to the process where plant roots uptake metal

contaminants from the soil and translocate them to their above soil tissues. As different

plant have different abilities to uptake and withstand high levels of pollutants many

different plants may be used. This is of particular importance on sites that have been

polluted with more than one type of metal contaminant. Hyperaccumulator plant species

(species which absorb higher amounts of pollutants than most other species) are used on

may sites due to their tolerance of relatively extreme levels of pollution.

Once the plants have grown and absorbed the metal pollutants they are harvested and

disposed of safely. This process is repeated several times to reduce contamination to

acceptable levels. In some cases it is possible to recycle the metals through a process

known as phytomining, though this is usually reserved for use with precious metals.

Metal compounds that have been successfully phytoextracted include zinc, copper, and

nickel, but there is promising research being completed on lead and chromium absorbing

plants. 

Rhizofiltration

Rhizofiltration is similar in concept to Phytoextraction but is concerned with the

remediation of contaminated groundwater rather than the remediation of polluted soils.

The contaminants are either adsorbed onto the root surface or are absorbed by the plant

roots. Plants used for rhizoliltration are not planted directly in situ but are acclimated to

the pollutant first. Plants are hydroponically grown in clean water rather than soil, until a

large root system has developed. Once a large root system is in place the water supply is

substituted for a polluted water supply to acclimatise the plant. After the plants become

acclimatised they are planted in the polluted area where the roots uptake the polluted

water and the contaminants along with it. As the roots become saturated they are

harvested and disposed of safely. Repeated treatments of the site can reduce pollution to

26

suitable levels as was exemplified in Chernobyl where sunflowers were grown in

radioactively contaminated pools.

Phytostabilisation

Phytostabilisation is the use of certain plants to immobilise soil and water

contaminants. Contaminant are absorbed and accumulated by roots, adsorbed onto the

roots, or precipitated in the rhizosphere. This reduces or even prevents the mobility of the

contaminants preventing migration into the groundwater or air, and also reduces the

bioavailibility of the contaminant thus preventing spread through the food chain. This

technique can alos be used to re-establish a plant community on sites that have been

denuded due to the high levels of metal contamination. Once a community of tolerant

species has been established the potential for wind erosion (and thus spread of the

pollutant) is reduced and leaching of the soil contaminants is also reduced. 

Phytoremediation of organic polluted sites

Phytodegradation (Phytotransformation)

Phytodegradation is the degradation or breakdown of organic contaminants by

internal and external metabolic processes driven by the plant. Ex planta metabolic

processes hydrolyse organic compounds into smaller units that can be absorbed by the

plant. Some contaminants can be absorbed by the plant and are then broken down by

plant enzymes. These smaller pollutant molecules may then be used as metabolites by the

plant as it grows, thus becoming incorporated into the plant tissues. Plant enzymes have

been identified that breakdown ammunition wastes, chlorinated solvents such as TCE

(Trichloroethane), and others which degrade organic herbicides.

27

Rhizodegradation

Rhizo-degradation (also called enhanced rhizo-sphere biodegradation, phyto-

stimulation, and plant assisted bioremediation) is the breakdown of organic contaminants

in the soil by soil dwelling microbes which is enhanced by the rhizo-sphere's presence.

Certain soil dwelling microbes digest organic pollutants such as fuels and solvents,

producing harmless products through a process known as Bioremediation. Plant root

exudates such as sugars, alcohols, and organic acids act as carbohydrate sources for the

soil micro-flora and enhance microbial growth and activity. Some of this compound may

also act as chemotactic signals for certain microbes. The plant roots also loosen the soil

and transport water to the rhizo-sphere thus additionally enhancing microbial activity. 

Phytovolatilization

Phyto-volatilization is the process where plants uptake contaminants which are

water soluble and release them into the atmosphere as they transpire the water. The

contaminant may become modified along the way, as the water travels along the plant's

vascular system from the roots to the leaves, whereby the contaminants evaporate

or volatilize into the air surrounding the plant. There are varying degrees of success with

plants as phyto-volatilizers with one study showing poplar trees to volatilize up to 90% of

the TCE they absorb. 

Hydraulic control of Pollutants

Hydraulic control is the term given to the use of plants to control the migration of

subsurface water through the rapid upltake of large volumes of water by the plants. The

plants are effectively acting as natural hydraulic pumps which when a dense root network

has been established near the water table can transpire up to 300 gallons of water per day.

This fact has been utilized to decrease the migration of contaminants from surface water

into the groundwater (below the water table) and drinking water supplies. There are two

such uses for plants:

Riparian corridors

28

Riparian corridors and buffer strips are the applications of many aspects of

phytoremediation along the banks of a river or the edges of groundwater plumes.

Pytodegradation, phytovolatilization, and rhizodegradation are used to control the spread

of contaminants and to remediate polluted sites. Riparian strips refer to these uses along

the banks of rivers and streams, whereas buffer strips are the use of such applications

along the perimeter of landfills.

Vegetative cover

Vegetative cover is the name given to the use of plants as a cover or cap growing

over landfill sites. The standard caps for such sites are usually plastic or clay. Plants used

in this manner are not only more aesthically pleasing they may also help to control

erosion, leaching of contaminants, and may also help to degrade the underlying landfill. 

Where has Phytoremediation Been Used?

As it is a relatively new technology phytoremediation is still mostly in it's testing stages

and as such has not been used in many places as a full scale application. However it has

bee tested successfully in many places around the world for many different contaminants.

This table shows the extent of testing across some sites in the USA

Location Application Pollutant Medium plant(s)

Ogden, UTPhytoextraction &

Rhizodegradation

Petroleum &

Hydrocarbons

Soil &

Groundwater

Alfalfa, poplar,

juniper, fescue

Anderson,

STPhytostabilisation Heavy Metals Soil

Hybrid poplar,

grasses

Ashtabula,

OHRhizofiltration Radionuclides Groundwater Sunflowers

Upton, NY Phytoextraction Radionuclides SoilIndian mustard,

cabbage

Milan, TN Phytodegradation Expolsives waste GroundwaterDuckweed,

parrotfeather

29

Amana, IARiparian corridor,

phytodegradationNitrates Groundwater Hybrid poplar

Pro's & Con's of Phytoremediation

As with most new technologies phytoremediation has many pro's and cons. When

compared to other more traditional methods of environmental remediation it becomes

clearer what the detailed advantages and disadvantages actually are.

Advantages of phytoremediation compared to classical remediation

It is more economically viable using the same tools and supplies as agriculture

It is less disruptive to the environment and does not involve waiting for new plant

communities to recolonise the site

Disposal sites are not needed

It is more likely to be accepted by the public as it is more aesthetically pleasing

then traditional methods

It avoids excavation and transport of polluted media thus reducing the risk of

spreading the contamination

It has the potential to treat sites polluted with more than one type of pollutant

Disadvantages of phytoremediation compared to classical remediation

It is dependant on the growing conditions required by the plant (ie climate,

geology, altitude, temperature)

Large scale operations require access to agricultural equipment and knowledge

Success is dependant on the tolerance of the plant to the pollutant

Contaminants collected in senescing tissues may be released back into the

environment in autumn

Contaminants may be collected in woody tissues used as fuel

30

Time taken to remediate sites far exceeds that of other technologies

Contaminant solubility may be increased leading to greater environmental

damage and the possibility of leaching

The low cost of phytoremediation (up to 1000 times cheaper than excavation and

reburial) is the main advantage of phytoremediation, however many of the pro's and cons

of phytoremediation applications depend greatly on the location of the polluted site, the

contaminants in question, and the application of phytoremediation. 

Phytoremediation & Biotechnology

The first goal in phytoremediation is to find a plant species which is resistant to or

tolerates a particular contaminant with a view to maximizing its potential for

phytoremediation. Resistant plants are usually located growing on soils with underlying

metal ores or on the boundary of polluted sites. Once a tolerant plant species has been

selected traditional breeding methods are used to optimize the tolerance of a species to a

particular contaminant. Agricultural methods such as the application of fertilisers,

chelators, and pH adjusters can be utilized to further improve the potential for

phytoremediation.

Genetic modification offers a new hope for phytoremediation as GM approaches can be

used to over express the enzymes involved in the existing plant metabolic pathways or to

introduce new pathways into plants. Richard Meagher and colleagues introduced a new

pathway into Arabidopsis to detoxify methyl-mercury, a common form of environmental

pollutant to elemental mercury which can be volatilised by the plant.

The genes originated in gram-negative bacteria

MerB encodes a protein organo mercuriallyase converts methyl mercury to ionic

mercury

MerA encodes mercuric reductase, which reduces ionic mercury to the elemental

form

31

Arabidopsis plants were transformed with either MerA or MerB coupled with a

consitutive 35S promoter

The MerA plants were more tolerant to ionic mercury, volatilised elemental

mercury, and were unaffected in their tolerance of methyl-lmercury

The MerB Plants were significantly more tolerant to methyl-lmercury and other

organomercurials and could also convert mthylmercury to ionic mercury which is

approximately 100 times less toxic to plants

MerA MerB double transgenics were produced in an F2 generation. These plants

not only showed a greater resistance to organic mercury when compared to the

MerA, MerB, and wildtype plants but also capable of volatilising mercury when

supplied with methylmercury.

The same MerA/MerB inserts have been used in other plant species including

tobacco(Nicotiana tabacum), yellow poplar(Liriodendron tulipifera).

Wetland species (bulrush and cat-tail) and water tolerant trees (willow and poplar)

have also been targeted for transformation.

3.3.Introduction of Phytoremedation

3.3.1 Start up of Phytoremedation

PROPERTIES OF DUCKWEED

The family lemnacae consists of two sub-families (Lemnoidea and Wolffioideae),

with four genera (Spirodella, Lemna, Wolffia and Wolffina), encompassing at least 34

species (Landolt, 1986). All plants are tiny (0.4 to15 mm) and identification is therefore

difficult (Leng, 1999).Duckweeds are mono cotyledonous, floating plants, and are the

32

world’s smallest and simplest flowering plants (Hillman and Culley, 1978). Each plant

consists of little more than two, poorly differentiated fronds, a combination of leaf and

stem. The tissue is composed principally of chlorenchymatous cells, separated by large

inter cellular spaces that provide buoyancy. The upper epidermis is cutinized and sheds

water. In Lemna and Spirodella the roots are believed to be adventitious, are only a small

proportion of overall plant weight and lack root hairs. The other two genera lack roots.

An important feature of the structure is the almost total lack of woody tissue .Members of

the Lemnacae family are found almost world wide, being absent only in the Polar

Regions and deserts.

Distribution of species is however, far from uniform with the Americas having

over 60% of recorded species, and Australia and Europe each having less than 30% of the

total. Species recorded in Australia comprise Spirodella polyrrhiza; S. punctata; Lemna

disperma; L. trisulca; L. aequinoctialis; Wolffia australiana; W. angusta (Landolt, 1986).

The habitat requirements of duckweed vary between species, but all share the need for

sheltered still water. Depth of the plant mat is an important limitation to growth. A

striking feature of duckweed species is their enormous reproductive capacity. Under

favorable conditions they have been reported as doubling their biomass every 16 to 48

hours (Leng, 1999). The main form of reproduction is vegetative, through the production

of “daughter” fronds that arise from one of two lateral pouches at the base of the frond.

Whilst vegetative growth is usual, duckweed daughter fronds do not stay attached

indefinitely, but rather break and form new colonies, only a few generations old. This

novel facility has led to the suggestion that duckweed growth could be considered

analogous to microbial growth (Hillman, 1961). Individual fronds have a relatively short

life span of 3 to 10 weeks when in the vegetative phase, depending on species,

reproductive rate and photoperiod (Landolt, 1986).

By this time, an original “mother” plant may have given rise to a clonal colony of

tens of thousands of personality plants over more than 50 generations. There appears to

be distinctive differences in longevity and mature size between generations (Landolt,

1986) that may be expressed as cyclicity in the growth pattern of a colony. One of the

significant attributes of duckweed is its ability to be used as a source of proteinaceous

food with a favorable profile of important amino acids (Rusoff et al., 1980)

33

GROWTH CONDITIONS FOR DUCKWEED

The growth of lemnacae may be nearly exponential, if carbon dioxide, light and

nutrient supplies are satisfactory. Discussion in this review is limited to the three major

plant macronutrients (nitrogen, phosphorus, potassium). Calcium and sulphur are not

generally considered to be limiting to growth (Landolt, 1986), whereas nitrogen and

phosphorus influence growth strongly and have an interactive effect.

Lemnacae are able to absorb nitrogen as ammonium, nitrate, nitrite, urea and

some amino acids, however the first two represent the main nitrogen source for most

species. Minimum, optimal, and toxic levels of nitrogen vary greatly between species

and geographic isolates and increasing light intensity is thought to elevate optimal

nitrogen requirements for growth. Of the species studied, L. miniscula has the lowest

(0.0016 mM/l) and an unclassified species of Lemna the highest (0.08 mM/l) minimum

requirement for nitrogen (Landolt, 1986). Similarly, the maximum tolerated level of

nitrogen varies from 30 mM/l (L. miniscula) to 450 mM/l for L. aequinoctialis (Landolt,

1986). The optimal recorded nitrogen requirement ranges from 0.01 mM/l for W.

colombia, up to 30 mM/l for S. polyrrhiza (Landolt, 1986). Duckweed’s requirement for

phosphorous, is variable (0.003-1.75 mM/l) between species as is seen for nitrogen

requirement, but appears unrelated to it (Landolt, 1986). Duckweed is reputedly able to

accumulate up to 1.5% of its weight as phosphorus in nutrient rich waters (Leng, 1999).

Between species differences are also evident for potassium, with requirements also being

influenced by light intensity.

FACTORS AFFECTING GROWTH AND COMPOSITION OF DUCKWEED.

There is a great deal of literature published on actual and potential yields of

duckweed (Culley and Epps, 1973; Hillman and Culley, 1978; Rusoff et al., 1980; Oran

et al., 1987; Leng, 1999; Chowdhury et al., 2000). Unfortunately, there is little data

available that records the interactions between genotype and environment. Many trials are

based on short-term yields in small containers, with theoretical yields extrapolated to a

34

per hectare per annum basis. Perhaps because of this, reported yields of duckweed vary

widely. A summary of reported yields assembled by Leng (1999) show yields ranging

from 2 to 183 t(DM)/ha/year. The extremely large range of recorded yields suggests that

making estimates of productivity based on results from short trials in laboratory-scale

vessels is of questionable value. Significant variances in growth have been demonstrated

between species and different geographic isolates of the same species (Bergman et al.,

2000). A composite picture of yields of l. gubba on different media is shown in Figure 1.

These published results on actual and potential yield of duckweed indicate a general lack

of agreement on the growth of these plants. There are a number of factors that may

mediate these apparently conflicting results. Quite apart from procedural differences

(such as different tank sizes, flow rate/retention times) there are numerous physico-

chemical differences that make establishment of equivalence, and thereby direct

comparison difficult. Time of year (and hence ambient temperature and day length),

latitude, and pH of growth media can all have a substantial influence on the physiology,

and thus the growth of the plant. There are many factors that influence growth, and the

value of drawing comparisons between trials conducted without similar protocols and

isolates, is also of limited value. Additionally, the levels of available nutrient, as well as

species differences, can strongly influence both the quantity and quality of material

produced. These differences may be interpreted in light of the existence of deficient,

optimal and toxic levels for nutrients. Nitrogen in particular, whilst being an essential

macronutrient, is toxic at high concentrations. Little interest has been shown in recent

times in establishing an optimum nutrient range for growth of duckweed despite

inconsistencies in published literature. Recent work (Bergman et al., 2000; Al-Nozaily,

2001) indicates that best growth is achieved where total nitrogen concentrations range

from 10 to 40 mg N/l. However this conflicts with the work of Caicedo et al. (2000), who

reported that growth rates of S. polyrhiza actually declined over a range of 3.5 to 100 mg

N/l. It has been demonstrated that lower (6 to 7) pH levels ameliorate the toxic effects of

nitrogen (McLay, 1976; Caicedo et al., 2000) and Al-Nozaily (2000) has suggested that

this may be because the low pH limits ionization of ammonia species, resulting in a low

proportion of ammonia in solution. The optimal nutrient profile for growth of duckweed

doesn’t necessarily produce the best quality of plant material in terms of protein content

35

and digestibility. Leng (1999) has suggested that optimal protein content will be

obtained where nitrogen is present at 60 mg N/l or greater. Early field observations by

Culley and Epps (1973) suggested that a strong positive relationship existed between high

levels of dissolved nutrients and plant characteristics, especially protein and digestibility.

Subsequently, several other researchers have reported positive relationships between

nutrient concentrations and dry matter yield, crude protein and phosphorous content

(Whitehead et al., 1987; Alaerts et al., 1996). In contrast, Bergman et al., (2000) found

little difference in dry matter (DM) yield and no difference in protein content in L. gibba

grown over a wide range of nutrient levels (52 to 176 mg N/l) In practice, the depth of

water required to grow duckweed will be determined by the purpose for which it is being

grown, as well as management considerations (Leng, 1999). Ponds of less than 0.5 m

depth may be subject to large diurnal temperature fluctuations. The greater the depth, the

less likely it is that plants will have full access to nutrients in the water column. Recently

it has been found that surface area, rather than depth, influences nitrogen removal in a

duckweed lagoon (Al-Nozaily et al., 2000).

3.3.2 Factor influencing startup process of phytoremadation.

36

3.3.3 Advantages & Disadvantages of phytoremadation.

Advantages of phytoremediation

It is more economically viable using the same tools and supplies as agriculture

It is less disruptive to the environment and does not involve waiting for new plant

communities to recolonise the site

Disposal sites are not needed

It is more likely to be accepted by the public as it is more aesthetically pleasing

then traditional methods

It avoids excavation and transport of polluted media thus reducing the risk of

spreading the contamination

It has the potential to treat sites polluted with more than one type of pollutant

Disadvantages of phytoremediation

It is dependant on the growing conditions required by the plant (ie climate,

geology, altitude, temperature)

Large scale operations require access to agricultural equipment and knowledge

Success is dependant on the tolerance of the plant to the pollutant

Contaminants collected in senescing tissues may be released back into the

environment in autumn

Contaminants may be collected in woody tissues used as fuel

Time taken to remediate sites far exceeds that of other technologies

37

Contaminant solubility may be increased leading to greater environmental

damage and the possibility of leaching.

3.3.7 Scope of phytoremadation by Lemna.

Now a days conventional sewage treatment plant have high construction cost,

energy and maintenance expenses and increasing labour costs, traditional wastewater

treatment systems are becoming an escalating financial burden for the communities and

industries that operate them. For many rural communities, the availability of low-cost

land has meant that more extensive, low-energy treatment processes can be a cost-

effective alternative, especially for final treatment of effluent.

Usefulness and a cultural preference for mechanical infrastructure. Queensland, in

particular, is climatically well positioned to take advantage of lagoon treatment systems

that use aquatic plants as productive ‘sinks’ for wastewater nutrients from a wide range of

sources. Of these, duckweed-based treatment systems offer the most promise.

The result is greater discharged effluent standards in terms of reduced total

suspended solids (TSS) and nutrients. Nutrients contained in phytoplankton are difficult

to harvest and are generally released back into the environment, whereas duckweed is

easily harvested, which results in direct removal of nutrients from the waste stream.

In addition, evaporation from the water surface is reduced in DWT systems

(Bonomo et al. 1997), Duckweed works to purify wastewater in collaboration with both

aerobic and anaerobic bacteria. Therefore, the duckweed plants themselves should be

considered as only one scomponent of a complete DWT system. Flow of nitrogenous

nutrients within a DWT system utilizing bacterial processing and uptake by duckweed

plants. Heterotrophic bacteria decompose organic waste matter into mineral components

— specifically forms of ammonia nitrogen and orthophosphates that are readily up-taken

by the duckweed plants. Bacterial decomposition consumes oxygen and can cause the

mid-water zone to become increasingly anoxic and the bottom of the lagoon to become

anaerobic, providing further zones for specialized bacterial processing of organic matter

and de-nitrification a 10cm surface layer remains aerobic due to atmospheric oxygen

transferred by duckweed roots.

38

DWT has great potential for renovating effluent from a wide variety of sources

including municipal sewage treatment plants, intensive livestock industries (including

aquaculture), abattoirs and food processing plants. The effectiveness of DWT depends on

a system design that facilitates the correct combination of organic loading rate, water

depth and hydraulic retention time. These will vary depending on the effluent source and

the level of pre-treatment.

Bacterial oxidisation of organic matter and nitrification are facilitated here, aided

by the additional surface area for biofilms provided by the duckweed roots and fronds.

Most researchers, however, suggest that efficiency gains using DWT are greater in

secondary and tertiary treatment of effluent where organic sludge has already been

removed or converted into simple organic and inorganic molecules that can be used

directly by duckweed (Alaerts et al. 1996; Caicedo et al. 2000; Smith and Moelyowati

2001; Dalu and Ndamba 2003). In the Burdekin, as with most communities in Australia,

primary sewage treatment infrastructure exists to remove solids. The problems currently

encountered with municipal wastewater treatment include difficulties in meeting TSS and

nutrient (Total N & P, ammonia) discharge regulations.

Average Total Nitrogen uptake (mg/L/day), uptake efficiency (percentage of

influent TN removed by the treatment) and duckweed biomass produced (g/m2/day) at

three Effluent Retention Times (E.R.T.). Data derived from Willett et al. (2003).

A dense duckweed mat has also been reported to decrease and control mosquito

larvae and odour in a wastewater body by providing an interface between the water and

air (Culley and Epps 1973; Iqbal 1999).

DWT has great potential for renovating effluent from a wide variety of sources

including municipal sewage treatment plants, intensive livestock industries (including

aquaculture), abattoirs and food processing plants. The effectiveness of DWT depends on

a system design that facilitates the correct combination of organic loading rate, water

depth and hydraulic retention time. These will vary depending on the effluent source and

the level of pre-treatment. In the case where raw sewage (human or livestock waste) is to

be processed, the primary treatment objective is to remove solids. Duckweed will

39

enhance primary treatment in these ponds by maintaining anaerobic conditions and

reducing odour nuisance (Skillicorn et al. 1993). Duckweed may need an acclimatisation

period to adapt to the very high N levels in raw agricultural wastewaters.

Most researchers, however, suggest that efficiency gains using DWT are greater

in secondary and tertiary treatment of effluent where organic sludge has already been

removed or converted into simple organic and inorganic molecules that can be used.In the

Burdekin, as with most communities in Australia, primary sewage treatment

infrastructure exists to remove solids. The problems currently encountered with

municipal wastewater treatment include difficulties in meeting TSS and nutrient (Total N

& P, ammonia) discharge regulations. Domestic wastewater does not contain significant

concentrations of toxins or heavy metals (Skillicorn et al. 1993), polishing zones may

simply be considered to be the latter reaches of a continuous duckweed treatment process.

3.3.8 Design consideration for phytoremadation

DWT system design principles

There is no single ‘off-the-shelf’ DWT package that will serve all purposes.

Requirements will vary depending on: the effluent source and volume; the level of pre-

treatment; the regulated discharge quotas that need to be met; prevailing climate and

financial considerations. Large-scale studies from both developing and western parts of

the world have been conducted using various DWT system designs and effluent sources,

but common recommended design features can be identified.

Plug-flow design

A plug-flow system is the most appropriate for secondary and tertiary effluent

treatment using DWT. A plug-flow system will ensure maximum contact between

wastewater and duckweed, and minimise the possibility of short-circuiting (Smith and

Moelyowati 2001). This will facilitate the incremental reduction of nutrients in the

wastewater. Plug-flow systems are also most efficient for pathogen removal (van der

Steen et al. 1999).

The basic unit of plug-flow systems is a shallow rectangular lagoon. The system

can operate singly or as a series of lagoons. The length/width ratio should be as large as

possible to encourage plug-flow conditions (Figure 2). Alaerts et al. (1996) recommend a

40

ratio greater than 38:1 although this is often difficult to achieve due to practical reasons

such as cost. Bonomo et al. (1997) suggest a length/width ratio higher than 10:1 will

suffice.

Figure 2. A plug-flow lagoon design, which prevents short-circuiting of flow

between inlet and outlet, is most appropriate for DWT.

Nutrient uptake

Since duckweed will be the major nutrient sink in these lagoons, a greater

biomass will inherently result in greater nutrient uptake. Greater biomass growth will

occur at higher nutrient concentrations (up to a tolerance limit), but as duckweed

incrementally reduces nutrients from the water, high biomass growth cannot be

maintained. Since the ultimate object of treatment is to reduce nutrient concentration,

duckweed starvation inevitably will occur at the latter stage in the treatment process.

In a plug-flow system, nutrient concentrations will be higher at the beginning of

the effluent stream and lower towards the end. This will facilitate a ‘farming’ zone (high

duckweed production/high nutrient uptake) and a ‘polishing’ zone (lower overall

duckweed growth/lower nutrient uptake). In the farming zone, where growth nutrients (N

& P) are plentiful, duckweed plants are predisposed to absorb them to the exclusion of

other elements present in the wastewater column (Skillicorn et al. 1993). In the polishing

zone, however, duckweed plants starved of N and P nutrients will scavenge for sustaining

nutrients. In the process they can absorb toxins and heavy metals if present in the

InletEffluent flowDischarge wastewater. This will have implications on the reuse or

disposal of the harvested plants. However, since most agricultural or domestic

wastewater does not contain significant concentrations of toxins or heavy metals

(Skillicorn et al. 1993), polishing zones may simply be considered to be the latter reaches

of a continuous duckweed treatment process.

Uptake efficiency

The nutrient uptake efficiency (i.e. the percentage of influent nutrient removed by

the treatment) will be determined by the hydraulic retention time. While a short retention

time will maintain high nutrient levels (and therefore extend the ‘farming’ zone), the

overall percentage of nutrients removed from the effluent stream is lower. Conversely, a

longer retention period will result in a greater percentage of nutrients being removed, but

41

create a relatively less productive ‘polishing’ zone when nutrients become limiting. For

example, the Burdekin pilot trial (Willett et al. 2003) tested three effluent retention times,

i.e. 3.5 days, 5.5 days and 10.4 days. The relationship between total nitrogen (TN)

uptake, uptake efficiency and biomass production by DWT at different retention times

from this trial are given in Table 1.

Table 1. Average Total Nitrogen uptake (mg/L/day), uptake efficiency

(percentage of influent TN removed by the treatment) and duckweed biomass produced

(g/m2/day) at three Effluent Retention Times (E.R.T.). Data derived from Willett et al.

(2003).

Overall retention time required in a DWT system will vary depending on a range

of factors including the influent nutrient levels, temperature and the discharge standards

that must be met. In general,

20 days hydraulic retention time would appear to be a minimum guideline for DWT to

achieve acceptable discharge standards and pathogen reduction in municipal sewage

treatment (Skillicorn et al. 1993).

Retention time is in turn, a function of water depth and flow rate. Shallow ponds

are better than deep ponds, but the trade off is the increased land area required and the

lack of temperature buffering with shallow ponds. Water depths between 0.6m and 1.5m

have been suggested as the most suitable for large-scale DWT systems (Skillicorn et al.

1993; Smith and Moelyowati 2001). A horizontal plug-flow velocity up to 0.1m/sec will

prevent disturbance of the duckweed mat (Edward 1992). Therefore, based on the daily

volume of effluent to be treated, the required retention time, and the above plug-flow and

depth specifications, overall p

PROPERTIES OF DUCKWEED

The family lemnacae consists of two sub-families (Lemnoidea and Wolffioideae),

with four genera (Spirodella, Lemna, Wolffia and Wolffina), encompassing at least 34

species (Landolt, 1986). All plants are tiny (0.4 to15 mm) and identification is therefore

difficult (Leng, 1999).Duckweeds are mono cotyledonous, floating plants, and are the

world’s smallest and simplest flowering plants (Hillman and Culley, 1978). Each plant

42

consists of little more than two, poorly differentiated fronds, a combination of leaf and

stem. The tissue is composed principally of chlorenchymatous cells, separated by large

inter cellular spaces that provide buoyancy. The upper epidermis is cutinized and sheds

water. In Lemna and Spirodella the roots are believed to be adventitious, are only a small

proportion of overall plant weight and lack root hairs. The other two genera lack roots.

An important feature of the structure is the almost total lack of woody tissue .Members of

the Lemnacae family are found almost world wide, being absent only in the Polar

Regions and deserts.

Distribution of species is however, far from uniform with the Americas having

over 60% of recorded species, and Australia and Europe each having less than 30% of the

total. Species recorded in Australia comprise Spirodella polyrrhiza; S. punctata; Lemna

disperma; L. trisulca; L. aequinoctialis; Wolffia australiana; W. angusta (Landolt, 1986).

The habitat requirements of duckweed vary between species, but all share the need for

sheltered still water. Depth of the plant mat is an important limitation to growth. A

striking feature of duckweed species is their enormous reproductive capacity. Under

favorable conditions they have been reported as doubling their biomass every 16 to 48

hours (Leng, 1999). The main form of reproduction is vegetative, through the production

of “daughter” fronds that arise from one of two lateral pouches at the base of the frond.

Whilst vegetative growth is usual, duckweed daughter fronds do not stay attached

indefinitely, but rather break and form new colonies, only a few generations old. This

novel facility has led to the suggestion that duckweed growth could be considered

analogous to microbial growth (Hillman, 1961). Individual fronds have a relatively short

life span of 3 to 10 weeks when in the vegetative phase, depending on species,

reproductive rate and photoperiod (Landolt, 1986).

By this time, an original “mother” plant may have given rise to a clonal colony of

tens of thousands of personality plants over more than 50 generations. There appears to

be distinctive differences in longevity and mature size between generations (Landolt,

1986) that may be expressed as cyclicity in the growth pattern of a colony. One of the

significant attributes of duckweed is its ability to be used as a source of proteinaceous

food with a favorable profile of important amino acids (Rusoff et al., 1980)

43

GROWTH CONDITIONS FOR DUCKWEED

The growth of lemnacae may be nearly exponential, if carbon dioxide, light and

nutrient supplies are satisfactory. Discussion in this review is limited to the three major

plant macronutrients (nitrogen, phosphorus, potassium). Calcium and sulphur are not

generally considered to be limiting to growth (Landolt, 1986), whereas nitrogen and

phosphorus influence growth strongly and have an interactive effect.

Lemnacae are able to absorb nitrogen as ammonium, nitrate, nitrite, urea and

some amino acids, however the first two represent the main nitrogen source for most

species. Minimum, optimal, and toxic levels of nitrogen vary greatly between species

and geographic isolates and increasing light intensity is thought to elevate optimal

nitrogen requirements for growth. Of the species studied, L. miniscula has the lowest

(0.0016 mM/l) and an unclassified species of Lemna the highest (0.08 mM/l) minimum

requirement for nitrogen (Landolt, 1986). Similarly, the maximum tolerated level of

nitrogen varies from 30 mM/l (L. miniscula) to 450 mM/l for L. aequinoctialis (Landolt,

1986). The optimal recorded nitrogen requirement ranges from 0.01 mM/l for W.

colombia, up to 30 mM/l for S. polyrrhiza (Landolt, 1986). Duckweed’s requirement for

phosphorous, is variable (0.003-1.75 mM/l) between species as is seen for nitrogen

requirement, but appears unrelated to it (Landolt, 1986). Duckweed is reputedly able to

accumulate up to 1.5% of its weight as phosphorus in nutrient rich waters (Leng, 1999).

Between species differences are also evident for potassium, with requirements also being

influenced by light intensity.

FACTORS AFFECTING GROWTH AND COMPOSITION OF DUCKWEED.

There is a great deal of literature published on actual and potential yields of

duckweed (Culley and Epps, 1973; Hillman and Culley, 1978; Rusoff et al., 1980; Oran

et al., 1987; Leng, 1999; Chowdhury et al., 2000). Unfortunately, there is little data

available that records the interactions between genotype and environment. Many trials are

based on short-term yields in small containers, with theoretical yields extrapolated to a

per hectare per annum basis. Perhaps because of this, reported yields of duckweed vary

widely. A summary of reported yields assembled by Leng (1999) show yields ranging

from 2 to 183 t(DM)/ha/year. The extremely large range of recorded yields suggests that

44

making estimates of productivity based on results from short trials in laboratory-scale

vessels is of questionable value. Significant variances in growth have been demonstrated

between species and different geographic isolates of the same species (Bergman et al.,

2000). A composite picture of yields of l. gubba on different media is shown in Figure 1.

These published results on actual and potential yield of duckweed indicate a general lack

of agreement on the growth of these plants. There are a number of factors that may

mediate these apparently conflicting results. Quite apart from procedural differences

(such as different tank sizes, flow rate/retention times) there are numerous physico-

chemical differences that make establishment of equivalence, and thereby direct

comparison difficult. Time of year (and hence ambient temperature and day length),

latitude, and pH of growth media can all have a substantial influence on the physiology,

and thus the growth of the plant. There are many factors that influence growth, and the

value of drawing comparisons between trials conducted without similar protocols and

isolates, is also of limited value. Additionally, the levels of available nutrient, as well as

species differences, can strongly influence both the quantity and quality of material

produced. These differences may be interpreted in light of the existence of deficient,

optimal and toxic levels for nutrients. Nitrogen in particular, whilst being an essential

macronutrient, is toxic at high concentrations. Little interest has been shown in recent

times in establishing an optimum nutrient range for growth of duckweed despite

inconsistencies in published literature. Recent work (Bergman et al., 2000; Al-Nozaily,

2001) indicates that best growth is achieved where total nitrogen concentrations range

from 10 to 40 mg N/l. However this conflicts with the work of Caicedo et al. (2000), who

reported that growth rates of S. polyrhiza actually declined over a range of 3.5 to 100 mg

N/l. It has been demonstrated that lower (6 to 7) pH levels ameliorate the toxic effects of

nitrogen (McLay, 1976; Caicedo et al., 2000) and Al-Nozaily (2000) has suggested that

this may be because the low pH limits ionization of ammonia species, resulting in a low

proportion of ammonia in solution. The optimal nutrient profile for growth of duckweed

doesn’t necessarily produce the best quality of plant material in terms of protein content

and digestibility. Leng (1999) has suggested that optimal protein content will be

obtained where nitrogen is present at 60 mg N/l or greater. Early field observations by

Culley and Epps (1973) suggested that a strong positive relationship existed between high

45

levels of dissolved nutrients and plant characteristics, especially protein and digestibility.

Subsequently, several other researchers have reported positive relationships between

nutrient concentrations and dry matter yield, crude protein and phosphorous content

(Whitehead et al., 1987; Alaerts et al., 1996). In contrast, Bergman et al., (2000) found

little difference in dry matter (DM) yield and no difference in protein content in L. gibba

grown over a wide range of nutrient levels (52 to 176 mg N/l) In practice, the depth of

water required to grow duckweed will be determined by the purpose for which it is being

grown, as well as management considerations (Leng, 1999). Ponds of less than 0.5 m

depth may be subject to large diurnal temperature fluctuations. The greater the depth, the

less likely it is that plants will have full access to nutrients in the water column. Recently

it has been found that surface area, rather than depth, influences nitrogen removal in a

duckweed lagoon (Al-Nozaily et al., 2000).

APPLICATIONS

The ability of duckweed to sequester nitrogen and phosphorus, and in so doing

“cleanse” dirty water, has been widely discussed in the literature for nearly 30 years

(Culley and Epps, 1973; Hillman and Culley, 1978; Oran et al., 1986; Landolt and

Kandeler, 1987; Leng, 1999). Systems utilising various species of duckweed, either alone

, or in combination with other plants, have been used to treat primary and secondary

effluent in the U.S.A. (Zirschky and Reed, 1988), the Middle East (Oran et al., 1985) and

the Indian subcontinent (Skillicorn et al., 1993; van der Steen et al., 1998).

Notwithstanding this reputation, some species and isolates are apparently quite sensitive

to high levels of nitrogen and/or phosphorous (Bergman et al., 2000), and effluent with a

high biological oxygen demand (BOD), such as abattoir waste, may kill the plants.

Although duckweed has a reputation for absorbing large amounts of dissolved nitrogen,

the degree of absorption appears to vary with concentration of nitrogen, time, species,

and (at least in temperate zones) the season. There is also strong evidence that there is a

symbiotic, or at least a synergistic relationship between duckweed and bacteria, both in

the fixation of nitrogen (Duong and Tiedje, 1985), and the removal of Chemical Oxygen

Demand (COD) (Korner et al., 1998) from water.

Differences in methodology, scale, and the parameters, both recorded and

measured, make direct comparisons between the many trials in published literature

46

difficult. However most research indicates that duckweed removes 40 to 60% of nitrogen

in solution over a 12 to 24 day period. Volatilization may account for a similar loss of

nitrogen (Vermaat and Haniff, 1998), although recent work completed in Israel (Van der

Steen et al., 1998), has suggested that direct duckweed absorption may account for less

than 20% of nitrogen loss, and volatilization/ denitrification may account for over 70%

In a similar fashion, lemnacae are generally able to absorb 30 to 50% of dissolved

phosphorous, although one researcher (Alaerts et al., 1996) has claimed over 90%

removal in a working, full scale system.

Phosphorous uptake (as measured by tissue phosphorous) and crude protein,

increased linearly with increases in nutrient concentration, up to approximately 1.5 g P/l,

and increased in absolute terms, up to 2.1 g P/l (Sutton and Ornes, 1975). This was

recorded in conjunction with a proportional rise in nitrogen concentration, thus the

association between nitrogen and phosphorous concentrations was unclear. COD is a

measure that quantifies water quality as determined by dissolved oxygen. All research in

the use of duckweed for improving effluent quality has determined significant but

variable decreases in COD (Alaerts et al., 1996; Karpiscak et al., 1996; Bonomo et al.,

1997; Vermaatand Haniff, 1998; van der Steen et al., 1999). However, a substantial

decrease in COD would be expected in open ponds without the presence of duckweed

(Al-Nozaily et al., 2000), so this improvement may not be attributable to the actions of

duckweed. Simplistically, the duckweed’s environment is somewhat two-dimensional. In

practice, this means that once the surface of a body of water is completely covered, the

plant has limited further opportunities to grow. Thus, insituations where there are high

nutrient levels, the clearance of dissolved nutrients is likely to be limited by harvesting

rate.

The work of Whitehead et al. (1987) confirms that at high average nutrient levels

(short retention time), nitrogen and phosphorous removal is enhanced with increased

cropping rate, whereas low nutrient concentrations favour low cropping rates. This latter

state indicates that growth is limited by nutrient availability. Degradation of bacterial

pathogens is a complex process and a comprehensive discussion is beyond the scope of

the current paper. However, two groups conducting specific investigations into this

issue (Karpiscak et al., 1996; van der Steen et al., 1999) found that faecal coliforms

47

decreased by 50 to 90% and that Giardia and Cryptosporidium fell by over 80% in

eutrophic waters in which duckweed was grown.

48

4. Materials and methods

4.1. Cultures

Axenic stock cultures of Lemna minor L. were maintained on the Pirson±SeidelÕs

nutrient solution (Pirson and Seidel, 1950) and subcultured biweekly. The pH value of

nutrient solution was adjusted to 4.55 before autoclaving (120°C, 0.15 MPa, 20 min).

Experimental cultures were started by inoculating a healthy colony with 2±3 fronds from

stock cultures into the 100 ml Erlenmeyer ¯asks containing 60 ml of modi ®ed

HoaglandÕs nutrient solution (Krajn_ci_c and Devid_e, 1980) supplemented with

CaCl2, CaBr2 and their 1:1 mixture. Plants grown on modi®ed HoaglandÕs nutrient

solution without tested chemicals were used as control. The pH value of nutrient solution

was adjusted to 5.0. Both, the stock and experimental cultures were grown in chamber

conditions under 16 h photoperiod (¯uorescent light, 80 lE sÿ1 mÿ2) at 24 _ 2°C.

4.2. Tested chemicals

To investigate the in¯uence of high density brines saturated solutions of CaCl2 (q . 1300

g dmÿ3) and CaBr2 (q . 1610 g dmÿ3), as well as their 1:1 mixture, were added into the

modi®ed HoaglandÕs nutrient solution in volumes appropriate to achieve the following

concentrations: 0.025, 0.05, 0.075 and 0.1 mol dmÿ3. Atomic absorption

spectrophotometry (ASTM D 511- 93, 1995) and volumetric method (ASTM D 512-89,

1995) were used to determine an accurate amount of calcium chloride, calcium bromide

and some inorganic substances in these solutions (Table 1). Amounts of heavy metals

(Cd, Cr, Ni, V, Fe and Co) were under detectable levels. Detection limits for those

metals were (mg dmÿ3): Cd . 0:0005; Cr . 0:07; Ni . 0:008; V . 0:1; Fe . 0:005 and Co .

0:006 . Afterwards, we repeated the experiment by addition of CaCl2 _ 2H2O and CaBr2

49

of analytical grade (Sigma) into the modi®ed HoaglandÕs nutrient solution in amounts

appropriate to achieve the same concentrations of tested chemicals as before.

4.3. Lemna bioassay

Duckweed Lemna minor was exposed to tested solutions for two weeks. The

tested solutions on Lemna minor growth was evaluated due to the following end points

(1) Relative growth of frond number,

(2) Relative growth of fresh weight,

(3) Dry to fresh weight ratio,

(4) Relative covered by plants and

(5) Chlorophyll a and chlorophyll b content and their ratio.

Results obtained by evaluation of growth parameters were represented as mean

values of eight replicates. The control was represented as 100% and the results obtained

with treated plants were represented as percentage of control. Chemicals that affected

Lemna minor growth significantly different from each other and control were marked

with different letters. Experiment for determination of chlorophyll a and chlorophyll b

contents was repeated three times. Results were calculated as mean values and

represented as percentage of control.

In this study, the growth of duckweed was assessed in laboratory scale

experiments. They were fed with municipal wastewater at atmospheric temperature.

Temperature, DO, pH, TSS, TDS, Sulphate, Nitrate, Phosphate, BOD5, COD, total

nitrogen (TN), total phosphorus (TP) and ortho-phosphate (OP) removal efficiencies of

the reactors were monitored by sampling influent and effluent of the system. Removal

efficiency in this study reflects optimal results: 73-84% COD removal, 83-87% TN

removal, 70-85% TP removal and 83-95% OP removal. The results show that the

duckweed-based wastewater treatment is capable of treating the laboratory

wastewater.Wetland treatment process is a combination of all the unit operations in a

conventional treatment process plus other physico-chemical processes, sedimentation,

biological oxidation, nutrient incorporation, adsorption and inprecipitation. The use of

duckweed in low-cost and easy-to-operate wastewater treatment systems has been

50

studied because of rapid growth rates achieving high levels of nutrient removal. Whilst

low fiber and high protein content make it a valuable fodder. Duckweed is a small, free

floating aquatic plant belonging to Lemnaceae family. Duckweed is well known for its

high productivity and high protein content in temperate climates. They are green and

have a small size (1-3 mm).

Duckweed fronds grow in colonies that, in particular growing conditions, form a

dense and uniform surface mat .The reason for this is the rapid multiplication of duckweeds and high protein content of

its biomass. Duckweed wastewater treatment systems have been studied for a wide

range of wastewater types .In this study we have focused on nutrient removal efficiencies

and removal rates between 50-95% have been reported for duckweed covered systems. Indirect effects like provision of surface and substrate by bacterial growth, change of the

physicochemical environment in the water and the possibility of the direct removal of

small organic compounds by heterotrophic growth are discussed in the study. Aquatic

plant-based wastewater treatment lagoons are engineered systems in which aquatic

plants in association with bacteria can purify wastewater.Duckweed-covered sewage lagoons (DSL) removes organic matter primarily

through aerobic heterotrophic oxidation. For this it needs the active transportation of

oxygen into the liquid phase.

51

RESULTS AND DISCUSSION

Duckweed plant was inoculated into a primary treated sewage water systems for

aquatic treatment over 8 day’s retention time period to assess the plant’s efficiency in

improving physico-chemical, bacteriological and biological characteristics of sewage

52

water. The primary treated sewage water used in the experiment was taken from the

collector tank of the tertiary sewage water treatment plant.

Sr.No. Parameter Unit. Initial

concentration

2nd Day 4th Day 6th Day 8th Day % Decrease in

concentration

1 Temperature OC 29.4 23.4 22.5 20.6 24.2 17.69

2 pH OC 7.25 7.46 7.49 7.51 7.39 -1.93

3 DO OC 0.46 0.77 0.96 1.25 0.58 -26.09

4 TSS OC 379 28 20 16 14 96.31

5 EC   905 852 878 899 995 -9.94

6 TDS   579 545 559 578 637 -10.02

5 CO3 OC 0.1 0 0 0 0 100.00

6 HCO3 OC 268.6 265.9 244.5 239.4 308.7 -14.93

7 T alkalinity OC 268.6 265.9 244.5 239.4 308.7 -14.93

8 BOD OC 320       30 90.63

9 COD OC 800 159 130 111 88 89.00

10 Phosphorus OC 4.91 4.68 4.13 3.35 2.56 47.86

11 O

Phosphate

OC 1.5 1.49 1.45 1.423 0.534 64.40

12 Phosphate OC 11 10.5 9.25 8.12 6.2 43.64

13 Ammonia OC 10 6.5 4.7 2.2 2 80.00

14 Nitrate OC 8.32 1.8 0.5 0 0 100.00

15 Calcium   120 78 80 80 120 0.00

16 Magnesium   124.8 72 75 76.8 115.2 7.69

17 Sodium   69.7 68.85 70.6 73.95 76.5 -9.76

18 Cloride   197.82 156.9 159.3 161.6 181.1 8.45

19 Sulfate   150.33 109.9 102.6 97.3 128.6 14.45

Pysico-chemical parameter.

Data recorded in Table showed that, values of pH were always alkaline and

ranged between 7.25 as a minimum value recorded at zero days and 7.51 as maximum

value obtained after six days treatment period. A 7.5 pH was found to be the most ideal

for the successful establishment of a duckweed system and optimum pond performance.

Duckweed grew well at pH 6 - 7.5 with outer limits of 4 and 8. it has observed that

duckweed growth declines as the pH becomes more alkaline. The dissolved oxygen

values increased as temperatures values decreased, revealing that the more cooler the

water the more dissolved oxygen it can hold.

53

The sewage temperature is one of the crucial design parameters of duckweed

ponds. In the present experiment temperature ranged between 20.6oC and 29.4oC which

was within temperature tolerance limit for duckweed growth the upper temperature

tolerance limit for duckweed growth was around 34oC. Duckweed cold tolerance allows

it to be used for year–round wastewater treatment in areas where tropical macro phytes,

such as water hyacinths, can only grow in summer.

As evident from Table , total suspended solids (TSS) values decreased by

increasing treatment periods, reaching minimum concentration of 14 mg L-1 after 8 days

(reduced by 96.3%).

Data in Table revealed that total dissolved solids (TDS) recorded their minimum

values of 545 mg L-1, after two days treatment (TDS reduced by 5.9%) and then values

increased gradually to the end of the experiment reaching their maximum values of 637

mg L-1, after 8 days. showed that calcium (Ca), magnesium (Mg), sodium (Na) and

chloride (Cl) reached their minimum concentrations of 78, 72, 68.85 and 156.9 mg L-1,

respectively after two days, with a reduction percentage of 35%, 42%, 1.2% and 20.7%,

respectively and then their values returned to increase gradually till the end of the

experiment. On the other side sulfate concentrations showed a continuous gradual

removal by increasing retention time, where its values decreased from 150.33 mg L-1 at

zero days until reaching 97.3 mg L-1 after six days (reduced by 35.3%), then it increased

to reach 128.6 mg L-1 after 8 days.

Biochemical oxygen demand (BOD), chemical oxygen demand (COD),

phosphorus (P), ortho-phosphate, phosphate, ammonia (NH3 +) and nitrate (NO3 -)

showed a gradual removal by prolonged treatment periods (Table I). Data revealed that

duckweed mat effectively reduced BOD by 90.6% (reduced from 320 mg O2 L-1 at zero

days reaching 30 mg O2 L-1 after 8 days treatment), COD by 89% (reduced from 800

mg O2 L-1 to 88 mg O2 L-1), phosphorus by 48% (reduced from 4.91 mg L-1 to 2.56

mg L-1), orthophosphate by 64.4% (reduced from 1.5 mg L-1 to 0.534 mg L-1),

phosphate by 43.6% (reduced from 11.0 mg L-1 to 6.2 mg L-1), ammonia by 80%

(reduced from 10.0 mg L-1 to 2.0 mg L-1).

On the other side the present treatment conditions were capable of depleting the

water body of any detectable nitrates (NO3) after 6 days treatment period. The duckweed

54

contribution for the removal of organic material is due to their ability to direct use of

simple organic compounds. mentioned that duckweed significantly enhanced COD

removal in shallow batch systems.

Batch of 65 liters sewage a 30 - 50% reduction in phosphate, 56 - 80% reduction

in ammoniacal nitrogen and 66 - 80% reduction in BOD. Nitrogen uptake rates of fat

duckweed vary between 45 and 1670 mg N m2 d-1 while the direct contribution of

duckweed to P removal can vary between 9 and 61% Nitrogen and P removal by

duckweed uptake were mainly realized by newly grown tissue, not by increasing the

tissue N or P content that nitrogen removal was in the range of 50% - 75% and this range

for phosphate was 17% - 35% in the discharged duckweed treatment system. Total

alkalinity showed a continuous gradual removal by increasing retention time (Table).

Values decreased from 268.6 mg L-1 at zero days until reaching 239.4 mg L-1 after six

days (reduced by 10.9%), then it increased to reach 308.7 mg L-1 after 8 days.

The increase in total alkalinity recorded on the 8th day of the experiment might be

attributed to increased decomposition of organic matter, which in turn produced excess

CO2 in the water resulting in an increase of alkalinity concentration

Removal of heavy metals by duckweed aquatic treatment system.

The removal of heavy metals from primary treated sewage water All detected

heavy metals were progressively reduced after 8 days treatment period. Duckweed

aquatic treatment system performed 100% copper and lead removal after 8 days

treatment. The efficiency of duckweed aquatic treatment in heavy metals removal in

various water systems data obtained suggested a maximum reliability of systems.

Bacteriological parameters.

Data on efficiency of duckweed aquatic system in eliminating bacteria revealed

that total and fecal coliform counts decreased gradually with increasing treatment period

removal of fecal coliform in the range of 99.27% and 99.78%.

55

56

57

58

59

60

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Marija Vujevi_c received her B.Sc. degree in 1998 from the

University of Zagreb, Croatia. She is M.Sc. student at the

Faculty of Science, University of Zagreb. The subject of her

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_Zeljka Vidakovi_c-Cifrek received her B.Sc. (1990), M.Sc. (1993)

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67

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