CHAPTRE 1 INTRODUCTION

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.
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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 stems. Of not require structural tissue to support leaves and wastewater

these, applications of Lemna (duckweed) in

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 byproduct 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 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
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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 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.

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 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

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 of municipal 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 from municipal waste water. 5. Conducting batch studies to find optimum opreting condition of various parameters. TABLE 1.1

STANDRADS FOR WASTE WATER DISPOSAL

Sr.No.

Parameters Inland water surface Public sewers

Standards Land for irrigation Marine & costal area

Colour & odour

All efforts should be made to remove it as fact as possible 100 500 200 i)100 for process w.w. ii) 10% above for cooling water effect.

SS(mg/l)

3 4 5

pH Temperature Oil & grease (mg/l) 40 10 45 20

5.5 to 9.0 -10 45 At discharge 20

Total Nitrogen

100

--

--

100

7 8

BOD COD

30 250

350 --

100 --

100 250
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CHAPTRE 2 LITERATURE REVIEW

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 Influent COD BOD Total N NH3 Total P 500 50 40 17 6 Effluent 320 30 20 2 3 Elimination capacity % % 30-40 60 50 80-90 50 Moderate Good Good Excellent Good Remark

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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) ii) iii) B) C) D) Biological methods Physiochemical method Land application method

Reuse of wastewater or by product recovery Prevention of waste and waste strength reduction. Specific approach.

2.4. Process selection criteria for treatment of various domestic waste water.
Over the years, biological treatment has established as a costeffective 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 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.

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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 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 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 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 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 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.
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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 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. 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 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 duckweeds environment is somewhat two-

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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, in situations 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 decreased by 50 to 90% and that Giardia and Cryptosporidium fell by over 80% in eutrophic waters in which duckweed was grown.

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 Basrah Oil Refinery. Journal of Applied Phyto-technology 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.

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Lead (Pb),Copper (Cu),Cadimum (Cd) and Zinc (Zn) reached their minimum concentrations of 0.2,0.02,0.02and 12 mg. L , 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.
-1

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CHAPTRE 3 PHYTOREMEDATION
3.1. History & background
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). Environmental Protection Experts from the U.S.

Agency (EPA) and the Organization for

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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 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, duckweeds 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 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
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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., experimental studies have shown an

2009) in plants; several animal

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 Phytoextraction Rhizofiltration Organic Phytodegradation 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 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 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 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.

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 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
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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 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

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the world for many different contaminants. This table shows the extent of testing across some sites in the USA

Location Ogden, UT Anderson, ST Ashtabula, OH Upton, NY

Application

Pollutant

Medium Soil &

plant(s) Alfalfa, poplar,

Phytoextraction & Petroleum &

Rhizodegradation Hydrocarbons Groundwater juniper, fescue Phytostabilisation Heavy Metals Soil Hybrid poplar, grasses

Rhizofiltration

Radionuclides Groundwater Sunflowers Indian mustard, cabbage Duckweed, parrotfeather

Phytoextraction

Radionuclides Soil Expolsives waste Nitrates

Milan, TN Phytodegradation Amana, IA Riparian corridor, phytodegradation

Groundwater

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 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

3.5 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
Page 27

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. 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.

Page 28

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

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 methyllmercury

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.

Page 29

3.7.Introduction of Phytoremedation
3.7.1 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 worlds 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
Page 30

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 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
Page 31

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). 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

Page 32

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 laboratoryscale 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
Page 33

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.

Page 34

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).

3.8Advantages & Disadvantages Duckweed

The growth of duckweed is very fast about 16 times. Removal of BOD is 70-80%. Removal of BOD is 30-60%. Removal of heavy metals 95-99.99%. pH vary from 3-10 Removal of Coliform 90-99%. Reduction of mosquito in thick duckweed. 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.

3.9 Disadvantages of phytoremediation by duckweed (Lemna)

Temperature above 34oC may cause death plant. It is dependant on the growing conditions required by the plant (ie climate, geology, altitude, temperature).

Page 35

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 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, duckweedbased 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
Page 36

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 denitrification 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 pretreatment. 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
Page 37

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/m /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 pretreatment. In the case where raw sewage (human or livestock waste) is to be processed, the primary treatment objective is to remove solids. Duckweed will enhance primary treatment in these ponds by maintaining anaerobic conditions and reducing odour nuisance (Skillicorn et al. 1993). Duckweed
2

Page 38

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
Page 39

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
Page 40

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/m /day) at three Effluent Retention Times (E.R.T.). Data derived from Willett et al. (2003).
2

Page 41

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.

3.12 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 worlds 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
Page 42

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
Page 43

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 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). 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
Page 44

(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. 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 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),
Page 45

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 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,
Page 46

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).

3.15 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.
Page 47

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

Page 48

in the use of

duckweed for improving effluent quality has determined

significant but variable decreases in COD. 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-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, in situations 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 decreased by 50 to 90% and that Giardia and Cryptosporidium fell by over 80% in eutrophic waters in which duckweed was grown.

3.16 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

Page 49

to growth, 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.. 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.

There is a great deal of literature published on actual and potential 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 laboratoryPage 50

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.

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 ,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
Page 51

growth of duckweed doesnt necessarily produce the best quality of plant material in terms of protein content and digestibility. The optimal protein content will be obtained where nitrogen is present at 60 mg N/l or greater. The 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. In contrast little difference in dry matter (DM) yield and no difference in protein content in Duckweed 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. 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. 3.17.1 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.

Page 52

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 easyto-operate wastewater treatment systems has been 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
Page 53

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 conditions, form a dense and uniform surface mat . The reason for this is the rapid multiplication of duckweeds and high growing

protein content of its biomass. Duckweed wastewater treatment systems have been studied for a wide range of wastewater types .In this study we and removal rates between

have focused on nutrient removal efficiencies

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.

Page 54

CHAPTRE 5 MATERIALS AND METHODS

4.1 Treatment methodology

4.1.1Materials and Methods Experiments 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 from Gadchiroli 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
Page 55

, Sulfate, BOD5, COD, Oil and Grease , Phenols, Nitrates, Phosphates and some Heavy Metals before and after the experiment. The value before Phytoremediation 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 (%) in concentration 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 of waste 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 biotreatment 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.
Page 56

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 50 oC 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.

Page 57

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
Page 58

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

count, phytoplankton identification and counting and chlorophyll a determination.

4.2 Materials 4.2.1 Determination of duckweed growth rate: This was

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

Page 59

CHAPTRE 5 RESULUTS DESCUSSION

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 physicochemical, 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.
2nd Day 4th Day 6th Day 8th Day

AND

Sr.No.

Parameter

Unit.

Initial concentration

% Decrease in concentration

1 2 3 4 5 6 5 6 7 8 9 10

Temperature pH DO TSS
EC

29.4 7.25

23.4 7.46 0.77 28 852 545 0 265.9 265.9

22.5 7.49 0.96 20 878 559 0 244.5 244.5

20.6 7.51 1.25 16 899 578 0 239.4 239.4

24.2 7.39 0.58 14 995 637 0 308.7 308.7 30

17.69 -1.93 -26.09 96.31 -9.94 -10.02 100.00 -14.93 -14.93 90.63 89.00 47.86

mgO2/l Mg/lit Umhos/cm Mg/lit Mg/lit Mg/lit Mg/lit mgO2/lit mgO2/lit Mg/lit

0.46 379 905 579 0.1 268.6 268.6 320 800 4.91

TDS CO3 HCO3 T alkalinity BOD COD Phosphorus

159 4.68

130 4.13

111 3.35

88 2.56

Page 60

11

O Phosphate

Mg/lit

1.5

1.49

1.45

1.423

0.534

64.40

12 13 14 15 16 17 18 19

Phosphate Ammonia Nitrate Calcium Magnesium Sodium Cloride Sulfate

Mg/lit Mg/lit Mg/lit Mg/lit Mg/lit Mg/lit Mg/lit Mg/lit

11 10 8.32 120 124.8 69.7 197.82 150.33

10.5 6.5 1.8 78 72 68.85 156.9 109.9

9.25 4.7 0.5 80 75 70.6 159.3 102.6

8.12 2.2 0 80 76.8 73.95 161.6 97.3

6.2 2 0 120 115.2 76.5 181.1 128.6

43.64 80.00 100.00 0.00 7.69 -9.76 8.45 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. 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 yearround wastewater treatment in areas where tropical macro phytes, such as water hyacinths, can only grow in summer.

Page 61

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 L1), 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).

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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 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
Page 63

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%.

DISCUSSION
Data from the Duckweed batch plant found from Municipal Waste water treatment At Laboratory Experiment efficiency of plant successes to

TABLE 1.1 STAND. FOR WASTE WATER DISPOSAL Vis Vis ACTUL RESULT

Sr.No.

Parameters

Standards

After Phytoremadation by Duckweed

Inland water surface

Municipal Council Dispose this water at same site

Colour & odour

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

Moderate Colour & odour removed

2 3 4

SS(mg/l) pH Temperature

100 5.5 to 9.0 40

14 7.39 24.2
Page 64

Oil & grease (mg/l)

10

Nil

Total Nitrogen

100

Nil

7 8

BOD COD

30 250

30 88

Reach the standards up to Indian Water pollution control board. In future if Municipal Conical Gadchiroli needs to treat the waste water the authority is advised that the duckweed plant made of 1)RCC pond of size 4M X 20M X 0.30 M For every 23000 Lit of water. 2) Duckweed Culture required initially 15 Kg. for every 80 M2 3) pH of water should maintained 4-10 4) Temperature of water should maintained not more than 34o C 5) Flow Should Maintain o.5m/sec.

Main Feature Initial Cost of the Plant @ for 6 MLd is Rs 26 Lakh The cost of maintain of plant is Rs.2.75/ M3 . No power is required .

Page 65

CHAPTRE 6 COCLUSION

Lab scale Phytoremedation studies using Duckweed (Lemna) work conducted for treatment of domestic waste water study. The present study shown that Phytoremedation found highly Promising , Eco-friendly & low cost for treating domestic waste water & finds better alternative to

conventional technology for treating waste water at domestic level. Especially rural level based on the studies following conclusion drawn. Lab scale Phytoremedation studies using Duckweed (Lemna) work conducted for treatment of domestic waste water study.

1) 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
Page 66

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. 2) The dissolved oxygen values increased as temperatures values decreased, revealing that the more cooler the water the more dissolved oxygen it can hold. 3) 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. 4) 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 yearround wastewater treatment in areas where tropical macro phytes, such as water hyacinths, can only grow in summer. 5) 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%).

Page 67

6) 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, 7) 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. 8) 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. 9) Biochemical oxygen demand (BOD), chemical oxygen demand (COD), phosphorus (P), ortho-phosphate, nitrate (NO3 periods (Table). 10) Data revealed that duckweed mat effectively reduced BOD by 90.6% phosphate, ammonia (NH3 +) and

-) showed a gradual removal by prolonged treatment

(reduced from 320 mg O2 L-1 at zero days reaching 30 mg O2 L-1 after 8 days treatment), 11) 12) 13) 1) 14) 15) 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). 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-

Page 68

16)

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. 17) The duckweed 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. 18) Batch of 450liters sewage a 30 - 50% reduction in phosphate, 56 -

80% 19) 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 20) 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. 21) Total alkalinity showed a continuous gradual removal by increasing

retention time.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. 22) 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.

Page 69

Other Observations :
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, 1. The odur removal is considerably reduced from waste water. 2. Mosquito breeding is reduced moderately.

Page 70

Sr.No.

Parameter

Unit.

Initial concentration

2nd Day

4th Day

6th Day

8th Day

Temperature

29.4

23.4

22.5

20.6

24.2

Page 71

Sr.No.

Parameter

Unit.

Initial concentration 7.25

2nd Day

4th Day

6th Day

8th Day

pH

7.46

7.49

7.51

7.39

Page 72

Sr.No.

Parameter

Unit.

Initial concentration 0.46

2nd Day

4th Day

6th Day

8th Day

DO

mgO2/l

0.77

0.96

1.25

0.58

Page 73

Sr.No. 4 6

Parameter TSS TDS

Unit. Mg/lit Mg/lit

Initial concentration 379 579

2nd Day 28 545

4th Day 20 559

6th Day 16 578

8th Day 14 637

Page 74

Sr.No. 8 9

Parameter BOD COD

Unit. mgO2/lit mgO2/lit

Initial concentration 320 800

2nd Day

4th Day

6th Day

8th Day 30

159

130

111

88

Page 75

Sr.No. 10 11 12 13 14

Parameter Phosphorus O Phosphate Phosphate Ammonia Nitrate

Unit. Mg/lit Mg/lit Mg/lit Mg/lit Mg/lit

Initial concentration 4.91 1.5 11 10 8.32

2nd Day 4.68 1.49 10.5 6.5 1.8

4th Day 4.13 1.45 9.25 4.7 0.5

6th Day 3.35 1.423 8.12 2.2 0

8th Day 2.56 0.534 6.2 2 0

Page 76

Sr.No.

Parameter

Unit.

Initial concentration 120 124.8 69.7 197.82 150.33

2nd Day

4th Day

6th Day

8th Day

15 16 17 18 19

Calcium Magnesium Sodium Cloride Sulfate

Mg/lit Mg/lit Mg/lit Mg/lit Mg/lit

78 72 68.85 156.9 109.9

80 75 70.6 159.3 102.6

80 76.8 73.95 161.6 97.3

120 115.2 76.5 181.1 128.6

Page 77

Page 78

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Disposal and Reuse. Tata McGraw Hill Publication, New Delhi.

4. Abdullah, M.H. and B.Mustafa. 1999. Phreatic water quality

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Howard and T.T. Yamamoto. 2002. Nutrient removal fromswine lagoon liquid by Lemna minor. Am. Soc. Agric. Eng.,45:1003-1010.
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2000.
10.Lemna gibba growth and nutrient uptake in response to

different nutrient levels. In Animal, Agricultural and Food Processing Waste: Proceedings of 8th International

Symposium, October 9-11, 2000, Des Moines, Iowa.

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