Manoharbhai Patel Institute of Engineering and Technology

7/31/2019 Manoharbhai Patel Institute of Engineering and Technology

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1.1. GeneralThe 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 averageload 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.


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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.

The efficiency of duckweed (Lemna) 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 days retention time period under

local outdoor natural conditions. Samples were taken below duckweed

cover after every two days to assess the plants 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 ofLemna (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 macro-phyte 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


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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 malaria & other water born diseases. Heavy

Metals ,Oil and Grease ,Phenols, Sulphide, Sulphate ,Nitrate ,Phosphate,

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 are discharge in to aquatic bodies like river, ponds

and lakes, where it is posing a serious threat to the water quality andbecome 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


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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 to prevent light

entering except at the top, each tank with dimensions of 150 cm long,

100 cm wide and 30 cm deep and was filled with 450 L primary treated

sewage water. Duckweed (Lemna) plants ere collected from Gadchiroli

Municipal Waste Water drain. The stock were cleaned by tap water then

washed by distilled water inocula ofLemna plants were transferred to the

water systems for aquatic treatment. The experiment was kept under

outdoor local environmental conditions for eight days retention time.

1.4.1 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. Samples

volume taken every two days for each of phytoplankton count and

chlorophyll a determination was 100 ml.

Parameters measured. Physico-chemical analyses 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 and rechecked in


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laboratory using bench-top equipment to ensure data accuracy for

biological parameters including total coli form count and fecal coli-form

count, phytoplankton identification and counting and chlorophyll a

determination.

1.4.2 Determination of duckweed growth rate: This was

determined for fresh and dry weights. Samples of 20 cm2 areas ofLemna

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

Pytoremadation has many advantages: it can clean-up a wide range

of contaminants while also being cost-effective, natural, passive, and

aesthetic. Because views of trees and green space can also provide

important physiological and social benefits, phytoremadation has the

potential to treat more than on-site contamination; it may also help to

create stronger neighborhoods and industrial/business districts.

The objective of present research is to develop new natural plants

& micro-aquatic plats to remove pollutants presenting waste water &

investigate the techno-economic feasibility. Study also aims to determine

the optimum condition operating parameters.

1. Identification of plants & micro-aquatic plant for removal ofmunicipal waste water.

2. Fabrication of experimental setup.3. Fabrication of laboratory setup.


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4. Conducting batch studies for the removal of pollutants frommunicipal waste water.

5. Conducting batch studies to find optimum opreting conditionof various parameters.

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


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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 treatmentsi) Biological methodsii) Physiochemical methodiii) Land application method

B) Reuse of wastewater or by product recoveryC) 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).


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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

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 processselection. 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.


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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. 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.

The anaerobic treatment can also be divided into fixed and

dispersed growth processes. The dispersed growth system is also known

as anaerobic contact process and is similar to activated sludge except itdoes 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 process.

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


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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 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 FIXEDBED 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 thedispersed 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 per unit volume of

reactors. Therefore, they are a better choice whenever land is limited.


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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 andprocess 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.


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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.

They make anaerobic treatment economically feasible at lowambient 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. 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


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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/ de-nitrification 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 hasdetermined 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 duckweeds environment is somewhat two-


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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 ofL. 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, duckweeds potential as a bio-

indicator 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 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 speciesL. 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


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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-Boul 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

3.2.1.What is phytoremadation ?

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


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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 hyper-accumulators 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.

3.2.2 How Does It Work?


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Phytoremediation is actually a generic 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

3.3 Methods of Phytoremediation

Phytoremediation of metal contaminated sites

Phytoextraction (Phytoaccumulation)

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


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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 isconcerned 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 suitable levels as was exemplified in Chernobyl where sunflowers were

grown in radioactively contaminated pools.

Phytostabilisation


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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 plantametabolic 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.

Rhizodegradation:


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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 asBioremediation. 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 thusadditionally 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


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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 :

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 toremediate 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 orcap 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


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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,

UT

Phytoextraction &

Rhizodegradation

Petroleum &

Hydrocarbons

Soil &

Groundwater

Alfalfa, poplar,

juniper, fescue

Anderson,

STPhytostabilisation Heavy Metals Soil

Hybrid poplar,

grasses

Ashtabula,

OHRhizofiltration Radionuclides Groundwater Sunflowers

Upton,

NYPhytoextraction Radionuclides Soil

Indian mustard,

cabbage

Milan, TN PhytodegradationExpolsives

wasteGroundwater

Duckweed,

parrotfeather

Amana,

IA

Riparian 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.


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3.4 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 waitingfor 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 therisk of spreading the contamination

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

3.5 Disadvantages of phytoremediation

It is dependant on the growing conditions required by the plant (ieclimate, geology, altitude, temperature)

Large scale operations require access to agricultural equipment andknowledge

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


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Contaminants may be collected in woody tissues used as fuel 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.

3.6 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

forphytoremediation.

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. RichardMeagher 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.


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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

new 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)

3.7.2GROWTH CONDITIONS FOR DUCKWEED

The growth oflemnacae 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


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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 ofLemna 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). Duckweeds 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.

3.7.3 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


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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


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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

doesnt 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 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.


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Large scale operations require access to agricultural equipment andknowledge.

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 Contaminant solubility may be increased leading to greater

environmental damage and the possibility of leaching.

3.10 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


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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

componentsspecifically 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.

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 oxidization 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


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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.

3.11 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


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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 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.

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

between inlet and outlet, is most appropriate for DWT.

3.11.1Nutrient 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


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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.

3.11.2 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

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.

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).


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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


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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)

3.13 GROWTH CONDITIONS FOR DUCKWEED

The growth oflemnacae 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.








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). Duckweeds 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


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(Leng, 1999). Between species differences are also evident for potassium,

with requirements also being influenced by light intensity.

3.14 FACTORS AFFECTING GROWTH AND COMPOSITION OF

DUCKWEED.


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 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),


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

physiology, and thus the growth of the plant.












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 ofS.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 doesnt

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 levels of dissolved nutrients and

plant characteristics, especially protein and digestibility. Subsequently,


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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 inL. 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).

3.15 APPLICATIONS DUCKWEED

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.


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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


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to growth, whereas nitrogen and phosphorus influence growth strongly and

have an interactive effect.








requirement for nitrogen.. Duckweeds requirement for phosphorous, is

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

requirement, but appears unrelated to it . Duckweed is reputedly able to

accumulate up to 1.5% of its weight as phosphorus in nutrient rich waters.

Between species differences are also evident for potassium, with

requirements also being influenced by light intensity.

3.17 FACTORS AFFECTING GROWTH AND COMPOSITION OF

DUCKWEED.


yields of duckweed. 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.

The extremely large range of recorded yields suggests that making

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


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Page 51

scale vessels is of questionable value. Significant variances in growth have

been demonstrated between species and different geographic isolates of the

same species. 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.












concentrations range from 10 to 40 mg N/l.

However this conflicts ,that growth rates of duckweed 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 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


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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 in precipitation. The use of duckweed in low-cost and easy-

to-operate wastewater treatment systems has been studied because of rapid

growth rates achieving high levels of nutrient removal.Whilst low fiberand high protein content make it a valuable fodder. Duckweed is a small,

free floating aquatic plant belonging to Lemnaceae family. Duckweed is


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4.1 Treatment methodology

4.1.1Materials and MethodsExperiments were performed in a non-continuous batch for

Phytoremediation potential of duckweed (Lemna ) in the removal of

pollutants in Municipal waste water was determined in Laboratory

experiment.

4.1.2 Study area and samples collection:

Samples of wastewater and Lemna (duckweed) were collected in

June2012. The assignment was to reclaim the sewage water collected fromGadchiroli Muncipal area. The used water treatment project was started in

June2012 and finalized in July2012. The total area of bio-treatment pond is

1mX1.5mX0.3m and total storage capacity is 0.45m3 (450 lit.). Laboratory

plant are aerobic. The water and plant samples were collected in every two

days.

4.1.3 Plant sampling and analysis of waste water:

Municipal wastewater characteristics were determined by analyzing of

some Physicochemical parameters like water Temperature, pH , Total

Alkalinity, Turbidity, Total Suspended Solids, Total Dissolve Solids, Sulfide


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, Sulfate, BOD5, COD, Oil and Grease , Phenols, Nitrates, Phosphates and

some Heavy Metals before and after the experiment. The value beforePhytoremediation experiment was noted as initial value, while the value

recorded after the Phytoremediation experiment was indicated by final

value. Pollutants removal were considered as the reduction (%) inconcentration according to:

(A-B)/A 100%

A= Initial Concentration (before experiment) .

B=Final Concentration (after experiment) .Ducweed (Lemna) plants were collected to study the pattern ofwaste

water from municipal area of Gadchiroli and phytoremediation process for

sample. From each samples were collected in replicates. Plant samples were

put in clean plastic bags and labeled carefully by permanent marker. All the

collected plant samples were placed in newspapers for the absorption of

excessive water. After 24 hours plant samples were digested and filtered,

and volume rose to 100mL.

4.1.4 Wastewater sampling and physio-chemical analysis:

One and half-liter of water samples were collected from all bio-

treatment ponds. For sample collection the bottles were washed with hot

water followed by distilled water. During collection bottles were filled,

rinsed with the sample water 2-3 times, tightly capped and properly labeled.

Physical parameters of collected water samples were studied immediately,

which were collected in replicates from all the 7 bio-treatment ponds. In

physio-chemical analysis different physical parameters were studied. Colour

was determined by direct comparison with standards and presented in

somewhat arbitrary terms of colour scale, which was observed by naked eye.


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It was done during the sampling of water on the spot (Peavy et al., 1985).

Temperature was measured by using a mercury thermometer of 0oC to 50oC

range and with 0.2oC least count. The temperature of water samples was

measured on the spot. The pH of water samples was determined in

laboratory with pH meter. The conductivity of water samples was

determined in laboratory with the help of conductivity meter.

First of all the instruments were washed with distilled water and

rinsed with the water sample. Bulb was also washed with distilled water

before putting in each water sample. The same procedure was repeated for

all water samples. For the measurement of total dissolved solids (TDS) clean

china dishes were put into oven at 103 to 105C for dryness, which were

then cooled and weigh.

Filtered water samples (20mL) were put in china dish and placed in

oven at 103 to 105C for evaporation, later on cooled in desiccators and

weighed. The increase in weight of china dish gave the weight of dissolved

solids. The results are shown in mg/liter using the following formula:

TDS = Final weight of china dish-initial weight of china dish x1000

mL of water sample used

Water samples were collected in triplicates and nitric acid (HNO3)

was added in water samples after it in situ pH measurement. All the

collected samples of water(100mL) were filtered with the filtration assembly

using the filter paper nitrocellulose membrane diameter of 0.45 m. For the

analysis of water and plant samples atomic absorption was powered on and

warmed up for 30 minutes.


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After the heating of hollow cathode lamp, the air acetylene flame was

ignited and instrument was calibrated or standardized with different working

standards. By atomic absorption spectrophotometer heavy metals of each

water and plant sample were noted.

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 to prevent light entering except at the top,

each tank with dimensions of 150 cm long, 100 cm wide and 30 cm deep

and was filled with 450 L primary treated sewage water. Duckweed

(Lemna) plants ere collected from Gadchiroli Municipal Waste Water 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.

4.1.5 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. 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 and rechecked

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

biological parameters including total coli form count and fecal coli-form


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RESULTS

Duckweed plant was inoculated into a primary treated sewage water

systems for aquatic treatment over 8 days (2nd

July to 9th

July , 2012 )retention time period to assess the plants efficiency in improving physico-

chemical, bacteriological and biological characteristics of sewage 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 7.25 7.46 7.49 7.51 7.39 -1.93

3 DO mgO2/l 0.46 0.77 0.96 1.25 0.58 -26.09

4 TSS Mg/lit 379 28 20 16 14 96.31

5 EC Umhos/cm 905 852 878 899 995 -9.94

6 TDS Mg/lit 579 545 559 578 637 -10.02

5 CO3 Mg/lit 0.1 0 0 0 0 100.00

6 HCO3 Mg/lit 268.6 265.9 244.5 239.4 308.7 -14.93

7 T alkalinity Mg/lit 268.6 265.9 244.5 239.4 308.7 -14.93

8 BOD mgO2/lit 320 30 90.63

9 COD mgO2/lit 800 159 130 111 88 89.00

10 Phosphorus Mg/lit 4.91 4.68 4.13 3.35 2.56 47.86


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11 O

Phosphate

Mg/lit 1.5 1.49 1.45 1.423 0.534 64.40

12 Phosphate Mg/lit 11 10.5 9.25 8.12 6.2 43.64

13 Ammonia Mg/lit 10 6.5 4.7 2.2 2 80.00

14 Nitrate Mg/lit 8.32 1.8 0.5 0 0 100.0015 Calcium Mg/lit 120 78 80 80 120 0.00

16 Magnesium Mg/lit 124.8 72 75 76.8 115.2 7.69

17 Sodium Mg/lit 69.7 68.85 70.6 73.95 76.5 -9.76

18 Cloride Mg/lit 197.82 156.9 159.3 161.6 181.1 8.45

19 Sulfate Mg/lit 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 morecooler the water the more dissolved oxygen it can hold.

The sewage temperature is one of the crucial des

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