Chapter-1

Introduction

Aquaculture in Bangladesh has rapidly progressed in recent years with a contribution of 44% to the annual fish production. Among different techniques of aquaculture, polyculture is one of the most important techniques. The basic principle of fish polyculture systems rests on the idea that when compatible species of different feeding habits are cultured together in the same pond, the maximum utilization of all natural food sources takes place without harmful effects. Polyculture or mixed culture of carps has been found as an economically viable and technically sustainable in perennial water bodies (Alikhuni, 1957; Chen, 1976). The selection of fish species is very important for polyculture systems. In the present study, tilapia (Oreochromismossambicus), sharpunti (Barbadesgonionotus), grass carp (Ctenopharyngodonidella), catla (Catlacatla) and mrigal (Cirrhinusmrigala) were selected for polyculture. These species are suitable for low inputs culture system in small ponds and ditches for their quick growth and for maximum production within short period. Bangladesh has numerous seasonal water bodies in the form of shallow ponds, ditches, roadside canals, pits in rice fields, which retain water for 4- 6 months. The natural environment of Bangladesh is also suitable for growing these fish species, which can be cultured in both shallow seasonal ponds and deeper perennial ponds. Duckweed are small floating aquatic plantswhich are widely available in Bangladesh and consist of four genera viz., Lemna, Spirodela,Wolfiaand Wolfiellaamong which about 40 species have been identified and in Bangladesh Lemna minor species are available(Journey et al., 1991; Skillikornet al., 1993). Its scientific classification is given in table 1. One of the most interesting alternative feeds is a small, aquatic plant knownas duckweed. Duckweed is the common name used to refer to the aquatic plantfamily of Lemnacea. Lemnacea consists of five genera: Lemna, Spirodella, Landoltia,Wolffia, and Wolffiellawith over forty identified species. Distributed throughout thetemperate and tropical zones of the world, duckweed is among the smallest of the flowering plants in the world (Skillicorn et al., 1993). Duckweed is commonly foundfloating on the surface of ponds, lagoons, and many other large stagnant bodies of their digestibility and making

Page | 1 them an ideal feed source (Leng et al., 1996). Because duckweed is a small, floating plant, it will not survive in water moving faster than 0.3meters per second. Duckweed spreads among bodies of water through migratingaquatic birds and floods (Skillicorn et al., 1993).

Recentlyduckweed has been accepted as protein rich (40-45% of the dry weight) feed for fish (Landolt and Kandeler, 1987; Lenget al., 1995; Sahaet al., 1999). According to Porath and Agami (1977), the weight of grass carp could be tripled (from, 100 to 300 g) within 50 days when feeding a mixture of Lemnagibbaand Lemna minor. Duckweed protein has higher concentration of essential amino acids, lysine and methionine than most plant proteins and more closely resembles animal protein in that respect (Journey et al., 1991). In Bangladesh, many studies have been carried out on the use of duckweed as feed for fishes in monoculture (Kohinoor et al., 1993; Bornali, 2004; Haque, 2005; Uddin et al., 2007; Chowdhury et al., 2008). Their structural and functional features have been simplified by natural selection to only those necessary to survive in an aquatic environment. Many species may have hair-like rootlets which function as stability organs. Table 1: Scientific classification of Lemna minor

Kingdom Plantae Subkingdom Viridiplantae Infrakingdom Streptophyta Superdivision Embryophyta Division Tracheophyta Subdivision Spermatophytina Class Magnoliopsida Superorder Lilianae Order Alismatales Family Araceae Genus Lemna Species Lemna minor L. – common duckweed, least duckweed

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Duckweed species have shown characteristics that make duckweed-based systems (DBS) very attractive, not only for wastewater treatment but also for nutrient recovery. Due to these characteristics, DBS have an important potential for resource recovery (Culley and Epps, 1973; Mbagwu and Adeniji, 1988).

Grown duckweed can be a good resource of proteins, as well as starch, and can be utilized for the production of value-added products, such as animal feed and fuel ethanol.Meanwhile, duckweed can be removed by simple and low-cost harvesting technologies.Consequently, duckweed has been used in the treatment of household and agricultural wastewater, especially in the last two decades. Duckweed has shown characteristics that make duckweed-based systems very attractive for wastewater treatment and nutrient recovery from synthetic or real swine wastewaters. Ammonium uptake is critically important for the treatment of swine wastewater, inwhich ammonium is the primary form of nitrogen. Duckweed preferentially absorbs ammonia rather than nitratebecause nitrogen in ammonium form is transformed directlyto plant protein, rather than being assimilated and subsequently reduced as in the case of nitrate. Knowledge of nutrient uptake kinetics would be useful in clarifying the functions of aquatic plants in constructed wetlands. Hence, knowing the kinetic properties of duckweed is important not only for screening species with high absorbing capacity, but also for the nutrient uptake by plants growing with other species in natural and agricultural ecosystems.

A current focus is on promoting fish farming for protein supply, increase income and employment opportunity. For fish farming, feed is one of the most important factor. Duckweed is used an unconventional feed for fish. Plants of the family lemnacae, known colloquially as duckweed, have been used in Asian primary production systems for hundreds of years to produce animal feed (Leng,1999).Duckweed (Lemna minor) is a simple tiny water plant that grows very well on pond surfaces. Moreover, it has high protein and carotene contents (Bui Xuan Men et al., 1995). Duckweed has been used as a main protein supplement for pigs (Bui Hong Van et al.,1997) and ducks (Bui Xuan Men

Page | 3 et al.,1995; Nguyen DucAnh et al 1997b). Duckweedhas received research attention because of its high nutritive value, especially the high protein content and also because of its capacity to grow rapidly on nutrient-rich waste water and produce biomass rich in protein (Leng et al., 1995).When effectively managed in this way duckweeds yield 10-30 ton DM/ha/year containing up to 43% crude protein, 5% lipids and a highly digestible dry matter. Fish production can be stimulated by feeding duckweed to the extent that yields can be increased from a few hundred kilograms per hectare/year to 10 tonnes/ha/year. Duckweed grown under ideal conditions and harvested regularly will have a fiber content of 5 to 15percent and a protein content of 35 to 45 percent.

Nitrogen in particular,whilst being an essential macronutrient, is toxic at highconcentrations. Little interest has been shown in recenttimes in establishing an optimum nutrient range for growthof duckweed despite inconsistencies in published literature.Recent work (Bergman et al., 2000; Al-Nozaily, 2000)indicates that best growth is achieved where total nitrogenconcentrations range from 10 to 40 mg N/l. However thisconflicts with the work of Caicedo et al,. (2000), whoreported that growth rates of S. polyrhizaactuallydeclinedover a range of 3.5 to 100 mg N/l.

1.1 Objectives

The objectives of the current research are as follows:

1. To assess the growth performance and survival of duckweed 2. To evaluate the effects of nitrogen on duckweed growth. 3. To determine the effects of nitrogen on nutrient content of duckweed.

1.2 Scope of the study

It is hoped that the study will provide a better understanding of the potential ofduckweed- based systems in Bangladesh by low-cost natural purification process. It will improve fisheries industry, production of fish and fishery product manufacturer in Bangladesh.

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Chapter-2

Review of literature

Before conducting a research by following experimental procedures, it is important to have a look on the previously conducted research activities on the related topics. So a review of the literature relevant to the present research work has been given below:

2.1 Duckweed Duckweed is one of the smallest macrophytes on the planet. It is a monocot, an angiosperm, floats on water, and has one of the fastest growth rates of any of the macrophytescontributing to its ability to be high accumulators of nutrients such as phosphorus and trace metals (Mkandawireet al,. 2004; Mkandawire and Dudel 2005, 2007; Odjegba and Fasidi 2004; Olguínet al,. 2005; Wang et al.,2004). Its location between the air-water interfacemakes it simple to separate from the water and susceptible to accumulation of hydrophobic chemicals (Brain et al., 2004a; Reinhold et al., 2010). Duckweed (Lemnaminor ), one of the fastest growing plant with high protein content is mainly exploited in the Asian developing countries e.g. Malaysia, Thailand, Vietnam, Israel, Bangladesh and India. Interest concerning duckweed first surfaced in the scientific community in between the late 1960’s to the early 1970’s. Research involving duckweed originally began on two separate fronts: as a wastewater treatment system and as a feed resource. The Asian society has long been incorporating duckweed into their daily lives as a wet feed source for poultry and livestock, a human food source, and for wastewater treatment of both human and animal waste. Due to duckweed’s prevalent use in Asian society, most of the research still originates from Asian universities and field stations.

2.2 Characteristics and Common Species in Bangladesh Lemnacea(common name duckweed) grows naturally in almost every region on earth with a growing season of at least 5 months. Most studies involving duckweed take place in climates with 9 to 10 month growing seasons. Rarer are the duckweed studies taking place in regions like Cache Valley with only 5 to 7 month growing seasons (Culleyet al. ,1981). Duckweed is a plant which helps it to grow in the colder climates. Nonetheless,

Page | 5 since water freezes in the winter and duckweed floats on water, it does best in warmer climates. Of the four principal duckweed genera, three are found in Cache Valley; the three species from these genera in Cache Valley are all reported to be cold tolerant (Culleyet al,. 1981; Landolt 1986). The duckweed plants growing in Cache Valley, Utah, include Lemnaturionifera(or L. minor), Wolffia borealis, and Spirodelapolyrhizza. L. turioniferaand L. minor are difficult to distinguish and both may exist in Wellsville. Landolt (1986) cataloged L.turioniferain Cache Valley. Duckweed’s native presence in Cache Valley, its tolerance to temperate climates, and its fast growth rates make it promising for nutrient removal in wastewater treatment applications. However, it has also attracted the attention of scientists because of its apparent high potential as a feed resource for livestock (Skillicornet al., 1993; Leng, et al., 1994).

2.3 Morphology of Duckweed Duckweed is the common name given to the simplest and smallest flowering plant that grows ubiquitously on fresh or polluted water throughout the world. Duckweeds have great application in genetic or biochemical research. This has been more or less in the same way that drosophila (fruit flies) and bread mould have been used as inexpensive medium for genetic, morphological, and physiological and biochemical research. Duckweeds are small, fragile, free floating aquatic plants. However, at times they grow on mud or water that is only millimetres deep to water depths of 3 metres. They grow slowly where nutrient deficiencies occur or major imbalances in nutrients are apparent. Duckweeds belong to four genera; Lemna, Spirodela, WolfiaandWolffiella. About 40 species are known worldwide. All of the species have flattened minute, leaf like oval to round "fronds" from about 1 mm to less than 1 cm across. Some species develop root-like structures in open water which either stabilise the plant or assist it to obtain nutrients where these are in dilute concentrations.Lemnaspecies are intermediate size at 6 -8 nm. Compared with most plants, duckweed fronds have little fiber -as little as 5 percent in cultured plants -because they do not need structural tissue to support leaves or stems. As a result virtually all tissue is metabolically active and useful as a feed or food product.

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Duckweed is a convenient feed for fish. Its attributes are:

 It can be readily grown locally often in waste ponds that are polluted.  It can be fed fresh and since it floats, by judicious setting of the rates of application it may be totally used by fish.  It is used very efficiently by fish such as tilapia and carp but other species might well cope with duckweed as a component of the diet since it is particularly low in fiber and high in protein when grown under ideal conditions.

It is relatively inexpensive to produce or may be regarded to have no cost where the opportunity costs of family labour are not taken into consideration.

2.4 The plant and its habitat

L. turionifera, W. borealis and S. Polyrhizzaspecies produce resting buds called turions, or in the case of L. minor produce resting fronds. Turions refer to starch-enriched overwintering buds that can sink to the bottom sediment during winter conditions and emerge under warmer conditions, thus enabling them to survive freezing weather (Landolt 1986). Turions can also be produced in warm temperatures (25ºC) since they were observed in the reactors during this and other studies (Landolt and Kandeler 1987). Resting fronds enable L. minor to survive cold temperatures and ice; but unlike turions, they do not sink to the sediment layer, but rather remain suspended in the water column between the sediment and ice. Resting fronds and turions are less buoyant than normal fronds which enables them to exist below the water surface when ice forms (Landolt 1986).

2.5 Ecological impact of duckweed (L. minor) Fresh duckweed (and also the dried meal) is suited to intensive production of herbivorous fish (Gaigeret al., 1984) and duckweed is converted efficiently to liveweight gain by carp and tilapia (Hepher&Pruginin, 1979; Robinette et al., 1980; van Dyke & Sutton, 1977, Hassan & Edwards 1992, Skillicornet al., 1993).

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In nature, duckweed serves as food for many species of fishes and aquatic birds as well as for other small creatures associated with this epiphytic community are an assortment of insects, including beetles, flies, weevils and aphids which are in turn eaten by the birds and fishes. As it grows very fast, a dense cover of duckweed limits the supply of solar energy and C02 to the submerged aquatic flora and prevents the algal bloom formation (Pokomy and Rejmankova, 1983). Algal bloom severely affects aquatic life by reducing the dissolved oxygen levels. Duckweed shades water, keeps it cool and thus allows for more dissolved oxygen available to fauna and minimizes water loss through evaporation. Duckweed shows an allelopathic effect on mosquito larvae. Extracts of Lemna minor caused significant mortality in the larvae of AedesaegyptiL., a known vector of human diseases.The presence of L. minor interfered with egg oviposition by Culexpipienspipiens and was lethal to C. p. pipienslarvae at the first instar stage (Eidet al., 1992). Layers of L. minor excrete amino-acids and humic substances into the aquatic environment which can provide nutrients to other organisms such as bacteria, submerged flora and indirectly to snails, springtails, isopods (Asellussp.) and other microdetrivores (Thomas and Eaton, 1996). Dead and dying duckweed fronds fall to the bottom of the water column where their decay contributes organic matter, nitrogen, phosphorus, and other minerals to the benthos. In addition cyanobacteria residing in the phyllosphere of duckweed fronds can fix atmospheric nitrogen, providing a nitrogen input in oligotrophic environments (Tran and Tiedje, 1985).

2.6 Duckweed culture in Bangladesh In Bangladesh, duckweed technology development and integration of wastewater treatment and fish aquaculture has been first introduced by the NGO PRISM Bangladesh in 1989. In 1993, a full-scale system for wastewater treatment and duckweed-based fish culture was installed at the Kumudini Hospital Complex (KHC) in Mirzapur (Gijzen and Ikramullah, 1999). In a joint cooperation between IRE and PRISM, a study was performed on the full scale duckweed based treatment system at KHC in Bangladesh (Alaertset al., 1996). PRISM Bangladesh has been continuously involved in duckweed- based wastewater treatment, both in centralized systems as well as in small-scale village settings. PRISM Bangladesh is operating a duckweed-based wastewater treatment system

Page | 8 in Mirzapur Kumudini Hospital Complex (KHC), Tangail since 1989. Total capacity of the wastewater treatment system is 14 million litres, 2.4 hectares of land are being used for duckweed based wastewater treatment plant and duckweed fed aquaculture. Duckweed-fed fish from the Mirzapur experimental project have a clear quality edge in the 'local market,fresh, green duckweed contrasted favorably with manure and other less appealing inputs to a conventional pond fishery. The Mirzapur experimental program in Bangladesh represents the first effort to apply existing knowledge on duckweed growth and cultivation develoved practical farming system. Mirzapur duckweed/carp polyculture ponds are currently the most productive carp pond in Bangladesh. On the basis of the experimental results the duckweed-based wastewater treatment system of Mirzapur Kumudini Hospital Complex PRISM Bangladesh has replicated another two duckweed- based wastewater treatment systems with aquaculture in Khulna City Corporation area under their Community-based Urban Wastewater Treatment Project, a component of Sustainable Environmental Management Program (SEMP) of UNDP. The first wastewater treatment located in Agricultural Training Institute (ATI) campus, Daulatpur, Khulna, total land used 1.4 ha, system capacity 400,000 liter/day and user population is about 7500- 9000. Sewage-grown lemnacae has formed the basis for a large, ongoing commercial operation at Mirzapur in Bangladesh, and hasyielded impressive results over several years (Skillicorn etal., 1993).The combination of duckweed-based sewage treatment and aquaculture is hemg practiced at a small scale (3,500 capita) by NOD in Bangladesh (Gijzen and Ikramuilah, 1999) .

2.6 Growing duckweed and its nutritive value

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.

Duckweeds can double their mass in between 16 hours to 2 days under optimal nutrient availability, sunlight, and water temperature. This is faster than almost any other higher

Page | 9 plant. Under experimental conditions their production rate can approach an extrapolated 183 metric tonnes/ha/year of dry matter although yields are closer to 10-20 tons of DM/ha/year under real-world conditions. The growth pattern resembles the exponential growth of unicellular algae more than that of higher plants and this confers a high potential for production as a livestock feed resource.

Growth rates of duckweed colonies will be reduced by a variety of stresses: such as nutrient scarcity or imbalance; toxins; extremes of pH and temperature; crowding by overgrowth of the colony and competition from other plants for light and nutrients. However, when conditions are good, duckweed contains considerable protein, fat, starch and minerals which appear to be mobilised for biomass growth when nutrient concentrations fall below critical levels for growth. Duckweed grown on nutrient-rich water has a high concentration of trace minerals, K and P and pigments, particularly carotene and xanthophyll, that make duckweed meal an especially valuable supplement for poultry and other animals, and it provides a rich source of vitamins A and B for humans. Table 2: The composition of duckweed harvested from a natural water source (Leng et al. 1994)

Contents Dry Weight (%) Protein 6.8-45.0

Lipid 1.8-9.2

Crude fiber 5.7-16.2

Carbohydrate 14.1-43.6

Ash 12.0-27.6

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2.7 Suitable nutrient for duckweed growth Nitrogen in particular,whilst being an essential macronutrient, is toxic at highconcentrations. Little interest has been shown in recenttimes 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 60 mg N/l. However thisconflicts with the work of Caicedo et al. (2000), whoreported that growth rates of S. polyrhizaactually declined over a range of 3.5 to 100 mg N/l.Leng (1999) has suggested that optimal protein content will be obtained where nitrogen is present at 60 mg N/l or greater.

The growth of lemnacae may be nearly exponential, ifcarbon dioxide, light and nutrient supplies are satisfactor. 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 andphosphorus influence growth strongly and have anointer active 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 greatlybetween 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, themaximum tolerated level of nitrogen varies from 30 mM/l(L. miniscula) to 450 mM/l for L. aequinoctialis(Landolt,1986). The optimal recorded nitrogen requirement ranges from 0.01 mM/l for W. colombia, up to 30 mM/l for S. polyrrhiza (Landolt,1986).Duckweed’s requirement for phosphorous, is variable (0.003-1.75 mM/l) between species as is seen for nitrogen equirement, but appears unrelated to it (Landolt, 1986).

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2. 8 Effects of urea on duckweed: Urea is the most efficient form of nitrogen supply to terrestrial crops, but its volatility in water and its elevating effect on pH makes it problematic for hydroponic applications. Skillicornet al., (1991) suggested that 4.5 kg urea/ha/day were enough to sustain a yield of 1,000 kg/ha/day of fresh duckweed. Urea contains approximately 45 percent nitrogen and is themost commonly available and lowest cost nitrogenous fertilizer.When applied towaterwithapH above 7.0,nitrogen losses through ammonia volatilization canoften exceed 50 percent. For example, urea is applied to theduckweed crop in Bangladesh at the rate of 20 kilograms per hectareper day (kg/ha/day), which is equivalent to 9.0 kg/ha/day ofnitrogen. Assuming a 50 percent loss before the crop is able to utilizethe nitrogen, 4.5 kg/ha/day is then available to support growth.

2.9 Effects of Temperature on duckweed growth condition

Like all biological systems, duckweed prefers certain growth conditions. Maintenance of these conditions is important Inachieving both efficient plant growth and effective wastewater treatment. Bangladesh, with its tropical climate and ambient temperature range of 8 °C to 39°C during the winter and hot season, respectively, sustain year round natural growth of duckweed (Gijzen and Kondker, 1997). Most species however, appear to exhibit optimum growth between 20°C to 30°C (Landolt,1986). The effect of light is positively reinforced by increases in temperature from 12°C up to 30°C at least, andmost species maximise growth at around 9,000 lux (at 24°C) (Landolt, 1986, sourced from Docauer (1983)).

2.10 Use of duckweed in fish nutrition

A major limitation to fish farming is that meals high in protein with high biological value are expensive and often locally unavailable. Duckweeds grown on water with 10-30 mg

NH3-N/l have a high protein content (around 40%) of high biological value (Hillman and Cully 1978). Fresh duckweed is highly suited to intensive fish farming systems with relatively rapid water exchange for waste removal (Gaigheret al., 1984) and duckweed is

Page | 12 converted efficiently to liveweight by certain fish including carp and tilapia (Hepher and Pruginin 1979; Robinette et al.,1980; Van Dyke and Sutton 1977; Hassan and Edwards 1992).

2.11 Utilization of duckweed as fish feed supplement A report by Porath and Koton (1977) has stated that the weight of grass carp could be tripled from 100 g to 300 g in a span of only 50 days by feeding a mixture of Lemnagibbaand L. minor. In 1999, Fasakin and coworkers presented data to support the use of duckweed in tilapia diets, but not as the sole source of protein. They found that the most cost effective (cost/unit weight gain in fish) diet to be one with 30 % duckweed inclusion (Fasakinet al., 1999). Research conducted by Bairagiet al., (2002) compared fermented duckweed meal with raw duckweed meal as replacements for fish meal protein supplements fed to rohu fingerlings. Kalitaet al.,(2007) have also demonstrated that commercial exploitation of L.minorcan be useful for the formulation of cost-effective and balanced artificial fishfeeds. Utilization of up to 30 % duckweed to replace commercial fish meal have been suggested by suggested by Effionget al., (2009), whereas utilization of 50 % duckweed was found better and cost effective measure for supporting both growth and profitability for fish production (Tavares et al., 2008).

2.12 Harvesting The quantity and frequency of duckweed harvesting plays a major role in the treatment efficiency and nutritional value of the plants. Regular harvesting ensures that the accumulated nutrients or toxins are permanently removed from the system. Because younger plants show a better nutrient profile and higher growth rate than older plants, regular harvesting is important to maintain a healthy and productive crop. Laboratory results from Whitehead and Bulley (Reddy and Busk, 1985) revealed that under conditions of high nutrient loading, an increase in the cropping rate resulted in improved nutrient removal. The harvested duckweed may be used as a valuable fish or animal feed (Skillicornet al., 1993).

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Chapter-3

Materials and Methods

Methodology is an indispensable and integral part of any research. In a scientific research the acceptability of the results depends to a great intent on the appropriate methodology. This chapter deals with the methods that are followed and materials that are used to achieve the objectives of the study. The results may be erroneous for the use of imperfect methodology. In this study a scientific and logical methodology has been followed by the researcher. The present study was carried out to Chattogram Veterinary and Animal Sciences University by providing facility and maintaining optimum conditions for experimental procedures. In order to achieve the objectives of this research, the following steps were followed:

3.1 Materials: The materials of the conteiners, duckweed, water, urea fertilizer as a source nitrogen, aerator are used.

3.2 Collection of duckweed: In Bangladesh duckweed species, Lemna minor are available. Duckweed (Lemnaminor ) was collected from a natural pond in the area of khulshi, Chattogram.

3.3 Duckweed stock culture This species (2-3cm) were collected from a local area and acclimatized to the experimental condition for one month in a tank at rooftop before experiment start. During the adaptation, the parameter of water quality were examined such as pH, temperature.

3.4 Selected duckweed for experiment

The duckweed was collected in the next step from the experimental condition for treatment. Before collected the pre-culture of the plant growth was carried out in culture medium for 6 days without harvesting. The qualities of these duckweed also was considered to ensure good quality cell. The best duckweed was selected and repeatedly washed five times with 1% aqueous hypochlorite solution to eliminate bacteria, algae, and other undesired organisms before taking in the culturing conteiners. After preprocessing, duckweed was cultivated for 7 days through a kind of culture medium.

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3.5 Sources of Nitrogen The original composition of the Huttner media (Landolt, 1987) was modified such that ammonium nitrogen or urea was the only source of nitrogen (Vermaat and Hanif, 1998).So urea fertilizer was used as fertilizer which are available in our country.Where 46% nitrogen present. These fertilizers were bought from the local market.

Table 3: Nutrient ingredients used in experiment and energy provided by urea

Fertilizer name Nutrient ingredient Urea 46% Nitrogen

3.6 Set up the container

At first the materials such as plastic containers opaque walls, impervious to light, to avoid algae proliferation, nylon net, pipe, aerator etc. were collected in local market for setting up the containers for conducting ‘Duckweed’ culture. The sizes of the containers were 1feet × 0.8 feet. These containers were contained more than 5 liter water. All containers were tagged individually for proper application of treatments. Netting was done by nylon rope over the containers to prevent birds.

3.7 Water quality management The mat of duckweed floating on the surface of a pond heats up in the sun much faster than the water column below it. The temperature differential several centimeters below the mat can beas great as 30°C. As surface temperature rise above 33°C, duckweed shows signs of heat stress which, if unrelieved, can damage the colony. There are two basic approaches to relieve heat stress: (1) passive measures such as shading and (2) active processes such as aerator. So that netting was done by nylon rope over the containers to prevent birds and aerator setup for heat stress. The average water temperature was 25°C.

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3.8 Experimental design

The duckweeds were grown in sterile cultures for 7 days in series comprising different concentrations of nitrogen; the series with various N-form were grown for seven days. Experiments were carried out in 500 ml water containing containers. The statistical design used for the experiment was completely randomized design (CRD). For the continuation of experiment, 12 plastic containers were set in the rooftop. In first week experiment was started within 4 duckweed cells. After 2 days all treatments duckweed were death except treatment- T0 because of the hyper nutrient for 4 duckweed cells. Then next week experiment was continued within each treatment contains 20 duckweed cells. The experiment was conducted with four treatments and each treatment had three replications. The treatments were conducted for 7days. But for the validity results experiment was continued 2weeks.

Table4 :TheNutrientingredients with inclusion level:

Treatment Nutrient Fertilizer concentration Concentration(mg- N/l) (mg Urea) st 1 treatment- T0 0 0

nd 2 treatment- T1 30 326.08

rd 3 treatment- T2 60 650.00

th 4 treatment- T3 90 978.26

3.9 Growth performance by weight

The weight of the duckweed was measured using an electronic balance. The growth performances were determined by average weight. Not only average weight of duckweed was measured but also daily weight gain was recorded for determination of growth performance. Average cell weight of duckweed, daily weight gain were recorded on daily basis.

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Following formula was used:

Daily weight gain (mg) = (Current sampling of duckweed weight – Immediate previous sampling of duckweed weight)per treatment per day

Average weight gain (mg) = (Final sampling of duckweed weight –Initial sampling of duckweed weight) per treatment per day

3.10 Growth performance by cell number

Duckweed cell was counted daily basis and cell number was recorded for determination of which treatment cell number was increased or decreased

Data of average cell number increase of duckweed = (current sampling cell number- immediate previous sampling cell number) per treatment per day

Average weight gain (Final sampling of duckweed cell number –Initial sampling of duckweed cell number) per treatment per day

3.11 Estimation of specific growth rate (SGR)

Specific growth rate was calculated by following formula:

( ) SGR= ln 푓𝑖푛푎푙푤푒𝑖푔ℎ푡 −ln (𝑖푛𝑖푡𝑖푎푙푤푒𝑖푔ℎ푡)*100 푑푢푟푎푡𝑖표푛 𝑖푛 푑푎푦푠

3.12 pH and Temperature measurement

The pH and temperature were measured every day. The average pH during a particular day was assumed to be the average of the pH measured just before the pH adjustment, and the pH that was set. The average pH and temperature for the culturing period was calculated by taking the average of the daily average pH and temperature values, for the three duplicates. The pH and temperature range was defined as the range between the maximum and minimum daily average value.

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3.13 Analysis of proximate composition

After the completion of research period, all treatments duckweed were collected from each replication for analysis of proximate composition. Collected duckweed were weighted and kept in oven for further analysis. After 24 hours drying duckweed were collected from oven kept those in desiccator. Samples were prepared before analysis. Proximate composition includes protein, lipid, moisture. These tasks were done in “Nutrition laboratory” of Faculty of Fisheries. Protein were determined by Kjeldahlapparatus, lipid by soxhlet apparatus, ash content by muffle furnace and moisture content by hot air oven.

3.14 Statistical analysis

Statistical analyses were performed by using MS excel (Microsoft office excel-2007, USA) and IBM SPSS Statistics 23 Version.Values are expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’spost hoc test to assess statistically significant differences among the control and different treated values. Statistical significance was set at P < 0.05. All results were presented as mean values and standard deviations.

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Some pictures

Plate 1: Duckweed culture

Plate 2: Urea Fertilizer

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Plate 3: Plastic container set up for Duckweed Culture

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Plate 4: Adding Nylon Ropes over the cages

Plate 5: Weighing of urea fertilizer

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Plate 6: Duckweed culture media

Plate 7: Weighing of sample

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Plate 8: Preparation of sample for laboratory analysis

Plate 9 : Laboratory analysis

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Chapter-4

Results

Nitrogen has a wide range of effects on aquatic plant. An attempt has been undertaken in the present study to demonstrate the effects of nitrogen on duckweed culture.

4.1 Effects of nitrogen on duckweed average cell number increase

Cell number of duckweed was calculated on daily basis by subtracting of current sampling cell number by immediate previous sampling duckweed cell number (per treatment per day). (Figure 3).During stocking of duckweed, the average cell number of duckweed was 20.The data showed that the duckweed provided with ‘Treatment-2(T2) higher cell number when comparing with other treatments. But after 5 days duckweed cell number was reduced.

Cell number increase chart (per treatment per day)

58

53

48

43 T0 T1 38 T2 T3

33 average cell number increage number cell average

28

23 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Time

Figure 1: Average cell number increase chart (Day 1-7)

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4. 2 Effects of nitrogen on weight gain per day

Weight gain of duckweed was calculated on daily basis by subtracting of current sampling weight gain by immediate previous sampling duckweed weight gain per treatment per day. (Figure 2)The chart showed that the duckweed provided with

‘Treatment-2(T2) higherand even growth in terms of weight when comparing with other treatments

Average weight gain (per treatment per day)

0.6

0.5

0.4

0.3

0.2 Wight gain (mg) gain Wight 0.1

0 D- 1 D-2 D- 3 D-4 D-5 D-6 D-7 Time

T0 T1 T2 T3

Figure 2: Daily weight gain in mg (Day 1-7)

4.3Growth performance of Duckweed

Growth performances of duckweeds are summarized in Table 5. Among dietary treatment, significant differences (p<0.05) were observed for final weight gain, cell number and specific growth rate. Except SGR, growth parametersand cell number were significantly higher in duckweed with T2 treated fertilizerswere shown in Figure 3,4, 5.

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Table 5: Growth performance of duckweed

Treatment Weight Cell number Specific growth rate

a a T0 (30 mg- N/l ) 6.40 ± 0.52 49.00±7.93 5.45 ±1.46 (6.00-7.00) (40-55) (4.10-7.00)

a a T1(30 mg- N/l ) 6.73 ± 0.47 57.33±6.42 6.03 ±1.28 (6.20-7.11) (50 -62) (4.57 -7.00)

b b T2( 60 mg- N/l ) 8.06 ± 0.12 96.00±10.14 7.92 ± 0.82 (7.98-8.20) (85-105) (7.00-8.57)

a a T3( 90 mg- N/l ) 6.00 ± 0.20 46.33±5.50 5.03 ±1.83 (5.80-6.20) (40-50) (3.36-7.00)

Level of 0.001 0.00 0.135 significance

Values are means ± S.D. Within a row, means with the same letters are not

significantly different (P < 0.05).

4.4 Effects of Nitrogen on duckweed weight

Sampling of the experimental duckweed was done in regular daily basis where weights of duckweed were taken by weight machine. During stocking of duckweed, the average weight of duckweed was 4.5 mg. In final sampling, it showed that the average weight of each treatment such as T0, T1, T2 and T3 were (6.40± 0.52) mg, (6.73± 0.47) mg, (8.06 ± 0.12) mg, (6.00 ± 0.20) mgrespectively. The data showed that the duckweed provided with ‘Treatment-2(T2) higher and even growth in terms of weight when comparing with other treatments (Figure 3).

The weight of duckweed of T1, T2 and T3 were compared to the control group.Values accompanied by different letters are statistically significantly different (p < 0.05, n=4)

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b a a a a

Figure 3: Effects of Nitrogen on weight of duckweed (Mean ± SD)

after 7days. 4.5 Effects of Nitrogen on specific growth rate

The specific growth rate of duckweed of T1, T2 and T3 were compared to the control group.Values accompanied by different letters are statistically significantly different (p < 0.05, n=4) in figure 4.

T0 T1 T2 T3

Figure 4: Effects of Nitrogen on specific growth rate of duckweed.

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4.6 Effects of nitrogen on cell number of duckweed

The cell number of duckweed of T1, T2 and T3 were compared to the control group. Values accompanied by different letters are statistically significantly different (p < 0.05, n=4).In final sampling, it showed that the average cell number of each treatment such as

T0, T1, T2 and T3 were (49.00±7.93), (57.33±6.42), (96.00±10.14),and (46.33±5.50) respectively. The chart showed that the duckweed provided with ‘Treatment-2(T2) higher cell number comparing with other treatments (Figure 5).The treatment -2 is significance where 60 mg/l Nitrogen used.

b

a a a

Figure 5: Effects of nitrogen on cell number of duckweed (Mean ± SD)

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4.7Water Quality Parameters

The temperature and pH is important factor for duckweed culture.The pH of water exerts a profound effect on the growth of duckweed. Average rang of temperature and pH were monitored which in table 6

Table 6: pHand Temperature analysis:

Treatment Temperature (°C) pH

T0 (0mg- N/l) 25.57± 2.16 8.10 ± 0.20

T1( 30 mg- N/l) 25.56 ± 2.06 8.20 ± 0.40

T2( 60 mg- N/l) 25.56 ± 2.15 8.23 ± 0.52

T3( 90 mg- N/l) 25.56 ± 2.09 8.29 ± 0.52

4.8Proximate composition analysis of Duckweed

Proximate composition such as protein, lipid, moistureof experimental duckweedswere listed in table 7.

Table 7: Proximate composition of experimental duckweeds

Treatments Protein(%) Lipid(%) Moisture(%)

(T0) 27.83 1.41 68.85

(T1) 28.02 1.44 69.57

(T2) 31.03 1.84 64.95

(T3) 31.93 1.70 65.33

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Chapter-5

Discussion

The present study was conducted to know the potentials of Nitrogen for better performance duckweed culture and evaluated the nutrient content of duckweed.Nitrogen is very important nutrient for duckweed growth. Without Nitrogen, the growth of all varieties was severely affected. A number of changes have been documented in this present study such weight, cell number,specific growth rate proximate composition, pH, temperature etc During research period, daily samplings were done where 1st four sampling (Day 1 to

Day 4) showed lower growth rate and lower weight gain T0, T1 because of where T0 was controlled and T1 Nitrogen amount was used 30 mg-N/l amount. And 60 mg-N/l (T2), and

90 mg-N/l(T3) growth rate was high. But After 4 days duckweed highest growth rate was showed T2 but T3 growth rate was decrease and water quality was deteriorated for others algae bloom,using high amount Nitrogen. Due to fluctuation of temperature, low temperature, rainfall and weather change duckweed cell was death and reduced inhibiting growth. After completion of research work with Nitrogen , the fresh matter production(in weight basis) increased from the lower to the higher doses up to 60 mg/l (T2), following a quadratic type of relationship (figure 3).Highest weight was found in T2 (8.06± 0.12) treated duckweed than the T0 (6.40± 0.52), T1 (6.73± 0.47) and T3 (6.00 ± 0.20). In this study, the maximum uptake rate was achieved in the 60 mg-N/l.Highest growth (in weight basis) was recorded in T2and the lower in T3 (Figure 2). Within higher concentrations resulted invariably in death of all duckweed.

The growth rate of duckweed had significant difference (p<0.05) at theTN(Total

Nitrogen) concentrations in the growth solution. At N concentrations of 30 mg/l (T1) 60 mg/l (T2), and 90 mg/l (T3), the average growth rates were 6.21 mg,6.30 mg, 5.48 mg respectively.Nitrogen in particular,whilst being an essential macronutrient, is toxic at high concentrations. Recent work (Bergman et al., 2000; Al-Nozaily, 2001) indicates that best growth is achieved where total nitrogenconcentrations range from 10 to 60 mg N/l.

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However thisconflicts with the work of Caicedoet al., (2000), whoreported that growth rates of S. polyrhizaactually declinedover a range of 3.5 to 100 mg N/l. The cell number of duckweed ranged from 49.00±7.93, 50.00±6.42, 96.00±10.14,

46.33±5.50, in different doses of 30 mg-N/l amount. And 60 mg-N/l (T2), and 90 mg-N/l

(T3) Nitrogen respectively. Highest cell number was found in (T2) during culture period and the lowest cell number was found in (T3) (Figure 5). However, the fresh matter production gradually decreased with increased dose of Nitrogen. According to (Bergman et al.,2000; Al-Nozaily,2001)indicates that best growth is achieved where total nitrogenconcentrations range from 10 to 60 mg N/l. It’s stated that increasing the Nitrogen to more than 60 mg-N/l in medium did not increase the growth significantly, indicating a luxury consumption of Nitrogen and the cells of duckweed were reduced, decreased frond size, fragile fronds, browning of roots, and marked browning of dorsal and ventral lobes. Nitrogen-deficient plants also showed reduction in the number of cell.

Duckweed is very rich in proteins, essential amino acids, vitamins, potassium, ferrous, copper, magnesium, zinc etc. Leng (1999) has suggested that optimal protein content will be obtained where nitrogen is present at 60 mg N/l or greater (Table 7). Protein contents are found inT0(without nutrient),30 mg/l (T1), 60 mg/l (T2), and 90 mg/l (T3) duckweed 27.83%,28.02%,31.03% and 31.93% in respectively(Table 7). Highest protein percentage was found in (T2) and lowest value found in which was not treated with Nitrogen(control)

(T0). Leng (1999) has suggested that optimal protein content will be obtained where nitrogen is present at 60 mg N/l or greater which is near about the present study. Also nitrogen treated duckweed has greater percentage of lipid and moisture than the control duckweed culture. The pH of water exerts a profound effect on the growth of duckweed, influencing the uptake of nutrients and especially the nitrate:amonium ratio and species availability (Caicedoet al.,2000).Optimal growth occurs around neutralpH for both SpirodellaandLemnaspecies, (Leng, 1999)and at around pH 5 for Wolffia(McLay, 1976). The lowerlimit of pH for growth in most species is disputed withLandolt (1986) suggesting a pH of 3 and Leng (1999) a pHof 5.

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The growth rate of duckweed is the result of many temperature dependent and interrelated reactions, with photosynthesis and respiration not having the same temperature curves. Most species however, appear to exhibit optimum growth between 20°C to 30°C (Landolt, 1986). The effect of light is positively reinforced by increases in temperature from 12°C up to 30°C at least, and, most species maximise growth at around 9,000 lux (at 24°C) (Landolt, 1986, sourced from Docauer (1983)).The average

Temperature of each treatment such as T0, T1, T2 and T3 were 26.56±2.16. 26.56± 2.06, 26.56 ± 2.15 and 26.56 ± 2.09respectively(Table 6). Also the average pH of each treatment such as T0, T1, T2 and T3 were 8.10± 0.50, 8.20 ± 0.40, 8.23± 0.52 and 8.29± 0.52respectively(Table 7).It has been demonstrated that lower (6 to 7) pH levels ameliorate the toxic effects of nitrogen (McLay, 1976; Caicedoet al., 2000) and Al- Nozaily (2000) has suggestedthat this may be because the low pH limits ionization ofammonia species, resulting in a low proportion of ammoniain solution. That’s whyT3 duckweed cell became brow, fragile, and poor growth.

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Chapter-6

Conclusions

Throughout agricultural history, people have always experimented with novel and unconventional practices to better the production and economic aspects of their operations. With current concerns of waste and by-product under-management and mismanagement, the area of alternative feeds has found itself on the frontline of improving agriculture along with the environment. Asia has long used alternative feeds in its agricultural practices due to the poor economic status of its village farmers. Duckweed is a good food for the fish. Much of the research conducted on the feasibility of duckweed as a feed or feed supplement originates from Asia. The present study was conducted on the basis of analysis of effects on nitrogen on duckweed which utilized by fishes. The results of this research showing that nitrogen has potential role in the better growth performance (weight, weight gain,cell number increase, SGR), proximate composition analysis. So duckweed culture will get the popularity in the fish farming. In aquaculture much cost is feeding cost. To minimize this problem we can use unconventional feed ingredients such as duckweed which is one of the most available and cheap ingredient. This type of research work will add a new dimension to improve fisheries industry, production of fish in Bangladesh.

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Chapter-7

Recommendation and Future perspectives

According to this research work, the following recommendations may be done:

 It is relatively inexpensive to produce or may be regarded to have no cost where the opportunity costs of family labour are not taken into consideration.  It has long been evident that duckweed has the potential to become a major protein commodity.  It can be readily grown locally often in waste ponds that are polluted. If duckweed, grown on wastewater, is to be consideredas an animal feeding supplement in any meaningful way.  Since availability of space is a major constraint for implementation of such a duckweed culture.  Duckweed has not yet been accepted as a commercial crop.  The major problem has been the economics of desiccation.

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Appendices

Appendix 1: Average 7 days cell number data of the duckweed at different sampling

Day-0 Day-1 Day-2 Day-3 Day-4 Day-5 Day-6 Day 7 T Cell No. Cell No. cell No. Cell No. Cell No. Cell No. Cell No. Cell No. T0R1 20 25 27 35 34 41 58 55

T0R2 20 21 22 26 39 45 45 40

T0R3 20 23 26 34 47 55 50 52

T1R1 20 29 38 46 55 62 58 50

T1R2 20 24 34 43 49 59 64 60

T1R3 20 31 40 35 41 55 58 62 T R 20 29 42 68 91 90 101 98 2 1 T2R2 20 24 45 60 82 85 90 105

T2R3 20 31 40 58 68 78 70 85

T3R1 20 38 49 59 68 50 58 50

T3R2 20 28 51 60 65 58 49 40

T3R3 20 39 56 55 69 65 56 49

*TR= Treatment × Replication, Cell No.= Cell Number

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Appendix 2: Data of average cell number increase of duckweed (current sampling cell number- immediate previous sampling cell number) per treatment per day

Day-0 Day- 1 Day- 2 Day -3 Day- 4 Day -5 Day- 6 Da-y 7

T ACNI ACNI ACNI ACNI ACNI ACNI ACNI ACNI

T0 20 23 25 31.66 40 47 51 49

T1 20 24.33 28.66 35.33 47 54 51 47.33

T2 20 25.33 32.66 41 50.33 58.66 57.33 54

T3 20 28 37.33 42 48.33 57.66 54.33 46.33

*T =Treatment, ACNI = Average Cell Number Increase

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Appendix 3: Average 7 days Weight data of the duckweed at different sampling :

Day -0 Day-1 Day-2 Day-3 Day-4 Day-5 Day-6 Day-7 TR Weight Weight Weight Weight Weight Weight Weight Weight (mg) (mg) (mg) (mg) ( mg) (mg) (mg) (mg) T0R1 4.5 4.59 5.11 5.2 5.2 5.21 5.8 6.15 T0R2 4.5 4.76 4.76 5.1 5.5 5.6 6.2 6.5

T0R3 4.5 4.6 4.71 4.98 5 5.9 5.5 6

T1R1 4.5 4.76 4.81 5 5.15 5.55 6 6.15

T1R2 4.5 4.54 4.72 5.4 5.55 5.85 6 6.76

T1R3 4.5 4.71 4.88 5.2 5.75 6.15 6.2 6.8

T2R1 4.5 4.91 4.98 5.8 5.9 6.5 7 6

T2R2 4.5 4.75 4.9 5.2 5.58 5.9 6.8 6.8

T2R3 4.5 4.65 4.98 5.15 5.98 6.6 6.5 6.9

T3R1 4.5 5 6.1 6.5 6.9 7.4 7 7.1

T3R2 4.5 5.1 6.5 6.95 7.2 7.35 6.9 7.2

T3R3 4.5 4.9 6.15 7.1 7.4 7.65 7.1 7.2

*TR= Treatment × Replication

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Appendix 4: Weight gain (current sampling weight- immediate previous sampling weight) data of the duckweed at different days

Day-1 Day-2 Day-3 Day-4 Day-5 Day-6 Day-7 TR Wt gain(mg) Wt gain(mg) Wt gain(mg) Wt gain(mg) Wt gain(mg) Wt gain(mg) Wt gain(mg)

T0R1 0.09 0.52 0.09 0 0.01 0.59 0.35

T0R2 0.26 0 0.34 0.4 0.1 0.6 0.3

T0R3 0.1 0.11 0.27 0.02 0.9 -0.4 0.5

T1R1 0.26 0.05 0.19 0.15 0.4 0.45 0.15

T1R2 0.04 0.18 0.68 0.15 0.3 0.15 0.76

T1R3 0.21 0.17 0.32 0.55 0.4 0.05 0.6

T2R1 0.41 0.07 0.82 0.1 0.6 0.5 -1

T2R2 0.25 0.15 0.3 0.38 0.32 0.9 0

T2R3 0.15 0.33 0.17 0.83 0.62 -0.1 0.4

T3R1 0.5 1.1 0.4 0.4 0.5 -0.4 0.1

T3R2 0.6 1.4 0.45 0.25 0.15 -0.45 0.3

T3R3 0.4 1.25 0.95 0.3 0.25 -0.55 0.1

*TR= Treatment × Replication, wt = Wight gain

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Appendix 5: Average weight (mg) data of duckweed per treatment per day

T D-1 D-1 D-2 D-3 D- 4 D-5 D-6 D-7 T0 4.5 4.65 4.86 5.09 5.23 5.57 5.53 6.15

T1 4.5 4.65 4.76 5.02 5.21 5.68 5.62 6.21

T2 4.5 4.63 4.74 5.12 5.23 5.76 5.83 6.56

T3 4.5 4.67 4.80 5.2 5.48 5.42 5.41 5.20 *T= Treatment, D= Day

Appendix 6 :Data of average weight gain (Final sampling of duckweed weight –Initial sampling of duckeweedweight) per treatment per day

Day- 1 Day-2 Day- 3 Day-4 Day-5 Day-6 Day-7

T AWG AWG AWG AWG AWG AWG AWG

T0 0.15 0.21 0.23 0.14 0.33 0.26 0.38

T1 0.20 0.21 0.26 0.19 0.46 0.21 0.31

T2 0.13 0.11 0.38 0.31 0.51 0.43 0.47

T3 0.17 0.13 0.39 0.28 0.36 0.21 0.16

*T = Treatment,AWG = Average Weight Gain (mg)

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Brief biography of the author

MelyAkterLaily is a candidate for the degree of MS under the Department of Aquaculture, the Faculty of Fisheries, Chattogram Veterinary & Animal Sciences University (CVASU) Khulshi-4225, Chattogram, Bangladesh.She is from Narsingdi.She passed Secondary School Certificate Examination in 2010 from Narsingdi Govt. Girl’s High School, Narsingdi, followed by Higher Secondary Certificate Examination in 2012 from Narsingdi Govt. Collage, Narsingdi.She obtained her B.Sc Fisheries (Hons.) Degree in 2018 from the Faculty of Fisheries, Chattogram Veterinary & Animal Sciences University (CVASU), Chattogram, Bangladesh.

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