MATERIAL & METHOD MATERIALS AND METHODS Experiments were performed in a non-continuous batch reactor system with settled

domestic wastewater in vessels of 0.841.2 l with dierent depth, mixing intensity and wastewater strength. Two groups of experiments were conducted (Table 1). Group 1 experiments allowed rationalizing the number and design of the Group 2 experiments, in view of the long duration of each experiment. Group 1 experiments used medium-strength settled sewage with

CODtotal 1 250 mg/l and studied system performance for two extreme depths (10 and 95 cm) and mixing intensity (G = 0 and 58 s 1 [34.1 W/m3]). Results suggested that depth does not aect COD removal; it only alters the initial CODtotal surface load ls,t (approx. 250 [at 10 cm] and 2400 kg/ha [at 95 cm]) (with variable CODtotal mass input). In Group 2 experiments, depths of 30, 70 and 95 were chosen to dierentiate between the inuence of initial CODtotal concentrations (200500 mg/l) and initial ls,t

(approx. 14004400 kg CODtotal/ha). In experiments 2/1 and 2/2 comparable surface loadings were applied but at a dierent COD concentration. The range of power dissipation between 0.75 and 3.0 W/m3 for aerated pond design, enabling solids re-suspension (Arceivala, 1998) was translated into a velocity gradient G (s 1) and consequently into a degree of turbulence (Appendix A) (Camp and Stein, 1943). CODlt was typically used as a parameter to quantify organic matter, as it is less dependent on variable and less relevant amounts of particulate matter. The

CODlt/BOD5,total ratio of settled sewage was 1.3. CODtotal /BOD5,total was typically 1.6 for settled inuent and 2 for e.uent. To study in Group 1 the role of the duckweed in O2 supply, triplicate reactors with and without duckweed (control) were used in parallel. Control reactors were covered with non-transparent plastic sheets to simulate duckweed mat. pH and DO were measured regularly and algal growth was monitored visually. In Group 2, no control reactors were used. Duplicates were found to be statisti-

cally suciently precise (se averaging 03% both in the case of duplicates and triplicates). Municipal raw wastewater (collected from BerkelRodenrijs or TNO Delft treatment plants, both in The Netherlands) was settled for 24 h to remove settleable matter. L. gibba was chosen because it is available in the Middle East and Yemen, and is most resilient to wastewater conditions (Al-Nozaily and Alaerts, in prep.). Clones were collected from Delft city canals and placed in wastewater for two weeks prior to the experiments to

allow them to acclimatize. Healthy clones were selected and used to create full cover in the duckweed reactors. Depending on the situation, mixing occurred by magnetic stirring or rotating paddle. Energy input was calculated according to equation (A2) (Appendix A). In the Group 2 experiments, a rotating paddle was used based on work by Coulson and Richardson (1987). Disturbances of the duckweed cover were prevented by surrounding the rod on the surface with an immobile PVC ring. Vertical ba.es attached to the reactor wall halted the rotation of

the duckweed mat. To suppress algal growth, duckweed was rinsed thoroughly after each harvest before reintroduction into the reactor. In addition, 2 mgCuSO4/l was added as recommended by Edwards et al. (1992). This Cu concentration can be tolerated by heterotrophs (Bolton and Klein, 1961), while the relative growth rate (RGR) of L. gibba was found to be not noticeably aected. Light was maintained at 130210 mE/m2 s using HPIT 400W Hg lamps under a time regime of 18/6 h on/o. Ambient and

water temperatures were 21248C and 19248C, respectively, while humidity was 40210%. Liquid samples were collected at 5, 30 and 65 cm depth every 5 days. Loading rates and ``inuent'' concentrations pertain to the initial reactor content. ``E.uent'' values pertain to the liquid in the reactor at the end of the experiment. Duckweed was harvested every 5 days by netting the biomass, rinsing it with tap water and drying the biomass by paper tissue (wet weight). Thereafter the initial stock density (500 2 100 g wet wt/m2) was restored in

every reactor. The increment from each replicate was mixed, and 510 g wet wt was taken for further analysis. Evapotranspiration was measured and compensated by adding tap water daily. Temperature, redox potential, DO and pH were measured every 23 days, and light intensity was checked every 5 days. BOD5 was analyzed using the Winkler method. The COD analysis was based on the closed reux technique (acid destruction at 1508C for 2 h) and colorimetry at 600 nm (Perkin Elmer 550 S, US) (APHA, 1992). Filtered

samples were prepared by ltering over GF/C (1.2 mm pores) glass ber lter paper. The DO was measured by WTW OX 196 (Germany). Redox potential was measured by platinum electrode AG 9100 from Metrohm Herisau (Switzerland). The light intensity was measured at the surface level of the experimental reactors by using a LI-COR radiation sensor, type SB (UWQ 4681, Campbell Scientic Ltd, UK). Air humidity was measured daily with a hygrometer. SPSS software was used for statistical analysis. Com-

parisons among mean values were made by analysis of variance (one-way ANOVA), signicant ANOVAs were followed by mean comparisons using Tuky's honestly signicant dierence test. Statistical analyses are reported as signicant when PR0.05.

MATERIALS AND METHODS Phytoremediation potential of duckweed (Lemna minor L.) in the removal of pollutants in refinery waste water was determined in Laboratory experiment. Experiment

To study pollutants removal capacity of Lemna minor L. from refinery wastewater, laboratory experiment was conducted. 20 L of Basrah oil refinery wastewater were used, and putted in four glass aquariums (273934) cm after diluted with deionized water in a 1: 4 ratio. Lemna minor were collected from the pond which located at Basrah University and transferred to laboratory that cleaned by tap water then washed by distilled water ,and 100g of fresh biomass of duckweed was putted in three aquaria and the fourth aquarium served as control that contain wastewater only. The experiment was kept under laboratory conditions of temperature (252) and lighting (8 light: 16 dark),

physiochemical analysis have been conducted after 7 days and repeated four times during a month.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 Phytotechnology in Environmental Sanitation, 1 (4): 163-172. 165

Analysis of refinery wastewater characteristic Refinery wastewater characteristics were determined by analyzing of some Physicochemical parameters like water Temperature, pH , Total Alkalinity, Turbidity, Total Suspended Solids, Total Dissolve Solids, Sulfide , Sulfate, BOD5, COD, Oil and Grease , Phenols, Nitrates, Phosphates and some Heavy Metals(Lead , Copper, Cadmium and Zinc). 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. All the analysis were done as described by

[14] [15] [16] [17]. 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) . Statistical analysis ;SPSS Software program was used to compare among means.

2. Materials and methods 2.1. Crowding experiment The growth rate of L. minor was determined under laboratory conditions (23 8C, a 14-h

photoperiod and an irradiance of 180 mmol m_2 s_1 PAR). The plants were grown on a liquid medium based on Smart and Barko (1985), optimised for Lemnacea by Szabo et al. (2003). A series of different densities was used (5.5, 9.5, 90, 180 and 915 g dry weight (DW) m_2). For each treatment, six replicates were applied. The initial biomass was determined as fresh weight. The plants were placed in vertical cylinders (height 10 cm, diameter 5.9 cm), which were placed in 2-l aquaria filled with medium (Szabo et al., 2003).

After day 4, the cylinders were placed in a basin with fresh medium. After day 7, the experiment was stopped and the fresh weight and dry weight (24 h at 70 8C) of L. minor were measured. The initial dry weight was calculated using the dry weight to fresh weight ratio at the end of the experiment. The relative growth rate was calculated assuming exponential growth. 2.2. Model A simple model was constructed to describe the effect of crowding, temperature and nutrients: dB dt

 Br f T; B;N; P _ l B (1) 246 S.M. Driever et al. / Aquatic Botany 81 (2005) 245251 The variation in time of the biomass of L. minor (B in g DW m_2) was modelled as the function of the maximum growth rate (r). The gross production was modified by a limitation function ( f(T, B, N, P)), which was a function of air temperature (T), biomass (B) and nutrients (N and P). Furthermore, the production was corrected for the loss (l), which included mortality, predation and respiration. The limitation function ( f(T, B, N, P)) was defined as: f T; B;N; P

T _ Tmin Topt _ Tmin N N hN P P hP hB B hB (2) Temperature (T) limitation was assumed to be linear from the minimum temperature (Tmin, 5 8C) up to the optimum temperature (Topt, 26 8C) (Landolt, 1986; Landolt and Kandeler, 1987). Nutrient limitation of ammonia and nitrate (N) and orthophosphate (P)

were modelled as Monod-type functions, with the following half saturation values: hN = 0.04 mg N l_1 and hP = 0.05 mg P l_1 (Luond, 1980). The limiting effect of biomass was simply assumed to be another Monod-type function dependent on biomass B and with a half saturation hB, which was determined during this study. 2.3. Field observations Three ditches in the surroundings ofWageningen, The Netherlands, were selected. In all ditches L. minor was present at the start of the monitoring period. For a period of 9 weeks

(AprilJuly 2003) biomass was measured in approximately 14-days intervals using stratified sampling. The water surface of the ditch was divided by eye into three strata, i.e. 020%, 2180% and 81100% coverage. The strata were drawn on a map and within each stratum, 10 random sampling coordinates were drawn. The biomass in the stratum with 0 20% coverage was neglected. Duckweed was sampled using a method after McLay (1974). A 10 cm _ 10 cm gauze covered iron square was positioned horizontally under the L. minor cover. The square was

lifted up through the cover of plants. All biomass not accounting for L. minor was removed by hand and dry weight was determined (24 h at 70 8C). Water samples for chemical analyses of N and P were taken at the last three sampling dates. NNO3 _ + NNO2 _, NNH4 + and PPO4 3_ were analysed using a Technicon Autoanalyser II. Air temperature data were obtained from a nearby weather station.

Materials and Methods Principle of duckweed based wastewater treatment: Duckweed has the capability to purify wastewater in collabration with both aerobic and anaerobic bacteria. The duckweed mat, which fully covers the water surface, results in three zones. These are the aerobic zone, the anoxic zone and the aerobic zone (Skillicorn et al., 1993). In the aerobic zone, organic materials

are oxidised by aerobic bacteria using atmospheric oxygen transferred by duckweed roots (Tchobanoglous and Burton, 1991). Nitrification and denitrification takes place in anoxic zones, where organic nitrogen is decomposed by anoxic bacteria into ammonium and ortho-phosphate, which are intermediate products used as nutrients by the duckweed (Smith and Moelyowati, 1998). The system consists of two tanks in which Lemna minor L. has been grown. Tanks are formed in dimension with 40x20x15 cm. The surface area of each tank is 800 cm2. The water depth of the reactors is 8 cm. The effective volume of the tank is 6.4

litres. Tanks are put into pond with a dimension of 80x50x16 cm to regulate environment temperature. Water temperature in the pond was around 210.5oC which was measured using special thermometer (JAGER). Light has been supplied by a special lamp (OSRAM day light 18 W) during day times. During night, the lamp was switched off by a timer. The wastewater was supplied from the effluent water of Bursa west side municipal wastewater treatment system and Bursa organized industrial estate wastewater treatment system. Duckweed cultures: Duckweed (Lemna minor L.) was collected

from Susurluk stream in the area of Karacabey, Turkey and adapted to laboratory scale system in Department of Environmental Engineering, Uludag University. Journal of Environmental Biology April 2007, 28(2) 307-314 (2007) Triveni Enterprises, Lucknow (India) For personal use only Free paper downloaded from: www. jeb.co.in Commercial distribution of this copy is illegal Journal of Environmental Biology _April, 2007_ Nihan Ozengin and Ayse Elmaci Analytical methods : At the influent and effluent of both reactors total nitrogen (TN), total phosphorus (TP), Ortho phosphate

(PO4-P) and chemical oxygen demand (COD) parameters have been measured for all retention times. COD analysis have been performed according to standart methods (APHA, 1998), other analyses have been performed with measurement kits and analysed using Dr. Lange Lasa 2 plus model photometer. Natural environment of Lemna minor L. was 19oC and pH was 7.1. Therefore, in the experimental study natural environment conditions for Lemna minor L. were constituted. Waste water samples were taken every three day from wastewater treatment points. Experimental mechanism was maintained every

day. pH was measured in every analyse at the influent and effluent of the system. COD analysis was observed throughout three weeks. TN, TP, OP were observed analysis throughout ten days. In addition to these analyses, monitoring for municipal and industrial wastewater was performed interval samples were taken throughout 48 hr (at 0, 15, 30, 45, 60, 120, 240, 360, 480, 960, 1440, 2880 min.) to determine the best removal time. Time (Day) 2 4 6 8 10 12 14 16 18 20 COD Removal Efficiency % 0 20 40

60 80 100 Municipal Wastewater Industrial Wastewater Fig. 2: COD removal efficiencies of municipal and industrial wastewater Time (Day) 5 10 15 20 COD (mg/L) 0 100 200 300 400 500 600 Municipal Wastewater _ndustrial Wastewater

Fig. 1: Influent and effluent COD concentration of municipal and industrial wastewater COD (mg/l) 308 Journal of Environmental Biology _April, 2007_ Performance of duckweed (Lemna minor L.)

2 Material and Methods Aquatic macrophytes Pistia stratiotes L. (water lettuce) was selected to assess its heavy metal removal capacities for Cr and Co from water under laboratory conditions. Pistia stratiotes L. is a perennial freshwater weed spread across the world and carries its entire life cycle as freefloating plant, only the root system is completely submerged. This species takes up metals from water, produces an internal concentration several folds greater than their surroundings and shows much higher metalaccumulating capacity than non-hyper

Proceedings of the International Academy of Ecology and Environmental Sciences, 2012, 2(2):136-138 IAEES www.iaees.org accumulating terrestrial plants. Therefore this species was selected for present phytoremediation experiment. The metal concentration was measured with the help of atomic absorption spectrophotometer (AAS) model: AA 7000, SHIMADZU and the standard was prepared using standard metal solution of Inorganic Ventures.

MATERIALS AND METHODS

DUCKWEED L. minor 8627 is a geographic isolate originally collected from Denmark in the worldwide duckweed germplasm collection (Landolt, 1998). Routine sterile cultures of L. minor 8627 were maintained in Schenk and Hildebrandt (SH) medium (Schenk and Hildebrandt, 1972) with 1% sucrose as a carbon source in a growth chamber at a constant temperature of 23C. Lighting at a photosynthetic photon flux density of 40 _mol m2 s1 in a 16hour photoperiod per day was provided by widespectrum fluorescent tubes

(Bergmann et al., 2000a). The duckweed was cultured in the SH medium supplemented with 3% sucrose for two weeks prior to the transfer to the synthetic swine lagoon liquid (SAM) for in vitro tests. For field experiments, duckweed was first grown in the SH medium with 3% sucrose for two weeks, then in SAM containing 3% sucrose for two weeks, and bulked in a greenhouse using diluted (50%) swine lagoon liquid for six weeks. IN VITRO TESTS In order to determine the intrinsic dynamics of nutrient uptake from the synthetic medium (SAM) by the duckweed

and its growth in relation to nutrient concentrations in the medium, other environmental parameters, such as initial duckweed biomass, temperature, and light intensity, were maintained constant. The synthetic medium (SAM) was formulated to closely resemble the nutrient profile of typical swine lagoon liquid in North Carolina (Bergmann et al., 2000a). Nutrient concentrations of SAM used as the medium in the in vitro tests are listed in table 1. Batch tests on the rate of nutrient (N and P) uptake from SAM by L. minor 8627 were conducted using 300 mL (5.1

5.1 11.6 cm) polypropylene boxes (Magenta Corp., Table 1. Ion concentrations and pH of the buffered synthetic medium (SAM). Ion (mg/L): Buffered SAM[a] NH4N 336.0 P 23.80 K 483.6 Ca 496 Mg 39.6 Cl 301.8 Fe 7.896 S 274.9 Na 175.5 B 0.693 Mn 8.8 Zn 3.074 Cu 1.207 Mo 0.02

Co 0.025 pH 7.0 [a] Buffering was accomplished by addition of 1.5 g/L citric acid. Vol. 45(4): 10031010 1005 Chicago, Ill.) in a growth chamber. Each box had a surface area of 25.8 cm2 and contained 150 mL (5.8 cm deep in the box) of SAM. To prevent bacterial contamination, the medium was sterilized in an autoclave for 30 minutes at 121C prior to use. Four batch tests were conducted to test four dilutions of SAM (100%, 75%, 50%, and 25% strength). Each batch test consisted of 52 boxes containing a dilution of SAM. Of these

boxes, 39 contained duckweed cultures, corresponding to triplicate samples for 13 time points, while 13 control boxes did not have any duckweed. Each duckweed culture was initiated with approximately the same amount of L. minor 8627 to cover the whole surface area with a single layer of duckweed. All boxes were then placed in the 23C growth chamber with the same lighting condition as routine cultures. The medium and the duckweed in each box were mixed briefly every day to ensure uniform distribution. Because growing duckweed decreased the medium pH, it was

necessary to adjust the pH to approximately 7.0 with drops of 10 M NaOH. During each test, three duckweed cultures and one control were removed for destructive sampling at every 48 hours to monitor the nutrient level and duckweed growth. Each batch test lasted for 22 to 24 days. FIELD EXPERIMENTS In order to investigate nutrient removal from swine lagoon liquid by L. minor 8627 under natural climate conditions of North Carolina, field experiments were conducted using concrete outdoor tanks built adjacent to a swine waste

treatment lagoon at the Lake Wheeler Road Field Laboratory of North Carolina State University in Raleigh, North Carolina. Seasonal effect on duckweed growth and nutrient removal was determined. The performance of the duckweed on the real swine lagoon liquid at different initial nutrient concentrations was compared with that in the in vitro tests. Two outdoor tanks were partitioned into 8 cells with PVC wallboard partitions. Each cell was 122 cm deep and had a surface area of 152 84 cm. The water depth in each cell during the experiments was maintained at 91 cm by adding

tap water to compensate for evaporation and transpiration. The water volume and surface area of each cell were 1.16 m3 and 1.28 m2, respectively. Two batch experiments were performed during consecutive twomonth periods in 1999: one from late May through late July (referred to hereafter as the spring experiment), and the other from midAugust through midOctober (referred to hereafter as the fall experiment). Since our previous experimental results had indicated that fullstrength swine lagoon liquid did not allow healthy growth of L. minor 8627 in a

greenhouse (Bergmann et al., 2000a), four dilutions (approximately 50%, 33%, 25%, and 20%) of the swine lagoon liquid were prepared with tap water in duplicate cells. At the initiation of each test, 2 kg (wet weight) of seed duckweed, enough to cover the surface of a cell, were added to each cell. The wastewater and duckweed in each cell were gently stirred for about 5 minutes every day. Wastewater and duckweed samples were taken every week after stirring to monitor the nutrient level in each cell. The biomass production was monitored by harvesting

duckweed present on approximately 20% of the surface area of each cell every Monday, Wednesday, and Friday. Frequent harvesting also encouraged duckweed growth and helped maintain healthy culture. The wet weight of the harvested duckweed was determined with a field balance after excess water was removed with a paper towel. Wet duckweed samples were dried in an oven at 105C for about 12 hours to determine the dry weight. Temperature in the wastewater was monitored with a StowAway TidbiT temperature recorder (Onset Computer Corporation, Bourne, Mass.), and

the light intensity at the duckweed surface was recorded with a LICOR light meter (LICOR, Inc., Lincoln, Neb.). The temperature and light intensity data were recorded every 15 min with dataloggers. CHEMICAL ANALYSIS Samples from the media in both in vitro tests and field experiments were analyzed for TKN, ammonium nitrogen (NH4N), nitrate nitrogen (NO3N), TP, orthophosphate phosphorus (oPO4P), chemical oxygen demand (COD), total organic carbon (TOC), and pH. The moisture content of the duckweed was determined from the wet and dry weights.

The dried duckweed biomass was analyzed for TKN and TP contents for the samples from the field experiments. All chemical analyses were conducted in the Environmental Analysis Laboratory of the Biological and Agricultural Engineering Department at North Carolina State University using EPA Methods (EPA, 1983) and APHA Standard Methods (APHA, 1995).

2. Methods 2.1. Construct design, Lemna transformation, and endocellulase screening The E1 endoglucanase gene from A. cellulolyticus was modiWed by PCR ampliWcation using template plasmid pPMT4-5, which contains the entire coding region for the enzyme (Thomas et al., 1996). Primers were designed to incorporate XbaI and SacI recognition sequences at the termini to facilitate cloning. The forward primer 5_-

tctagATGtattggcacacgagcgg-3_ (RE site underlined, start codon capitalized) also introduced a start codon adjacent to the signal peptide splice junction, while the reverse primer (5_-gagctcgtccggattgttgggttc3_) should anneal in the 3_ untranslated region of the E1 gene and therefore rely on the A. cellulolyticus stop codon. The putative translational product should not have a signal peptide and accumulate cytosolically. The amino terminus of the recombinant protein should also be diVerent from the native mature protein in that the Wrst four amino acids Ala GlyGlyGly are

replaced with the amino terminal Met. The sequence of the 1.6Kb modiWed E1 gene fragment was conWrmed and the fragment was cloned in place of the _glucuronidase gene of the binary vector pBI121 (ClonTech, Palo Alto, CA) using XbaI and SacI. The resultant plasmid, pCel25IX should result in expression of the recombinant E1 gene, under control of the constitutive cauliXower mosaic virus (CaMV) 35S promoter and nopaline synthase terminator. Agrobacterium tumefaciens-mediated gene transformation of duckweed was performed as previously described by

Yamamoto et al. (2001) using kanamycin selection. Fifteen independent transgenic duckweed lines with stable kanamycin resistance were established. Genomic integration of multiple recombinant constructs was conWrmed with PCR and genomic Southern hybridization as previously described (Yamamoto et al., 2001). Carboxymethyl cellulose (CMC)degrading activities of the transgenic duckweed plants were initially screened using modiWed CMC plate assays (Sharrock, 1988; Geimba et al., 1999), where one frond from each line was pinched by forceps, placed on an agar plate

containing CMC (degree of substitutionD0.7, medium viscosity) (SigmaAldrich, St. Louis, MO) and incubated for 16 h at room temperature, 37 or 70 C before staining with Congo Red to detect zones of hydrolysis. Presence of CMC-degrading activity in the duckweed culture medium was similarly estimated by placing 10 _l medium in a small well on the CMC detection plate. One duckweed line with consistently high levels of CMCdegrading activity, Cel25IX-15, was used for detailed characterization of the recombinant E1 enzyme.

2868 Y. Sun et al. / Bioresource Technology 98 (2007) 28662872 2.2. Analysis of duckweed transformant Cel25IX-15 In an eVort to more accurately determine whether expression of the E1 protein had any adverse eVects on growth, the transgenic duckweed line Cel25IX-15 and the untransformed control were identically cultured for two weeks in the SH medium (Schenk and Hildebrandt, 1972) supplemented with 1% (w/v) sucrose under the 16-h light/8h dark cycle at 23 C. The number of plantlets and total fresh weight were determined for each Xask. Data were

averaged for 10 replications per experiment. Experiments were repeated three times with Mean calculated by Student NewmanKeuls multiple range test (P<0.1). For analysis of recombinant protein production, duckweed was grown and harvested, as above, washed three times in excess (i.e. approximately three times the original volume of medium) deionized water to remove medium residues, and then frozen in liquid nitrogen. Total soluble protein was extracted from the harvested duckweed by grinding the whole plant materials with a mortar and a pestle

with 5ml of ice-cold 50mM sodium citrate buVer (pH 4.8)/g duckweed. Insoluble material was then removed by centrifugation at 10,000g for 10min at 4 C. Protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin as a standard (Bio-Rad, Hercules, CA). The CMC-degrading activity of transgenic duckweed lines was quantiWed according to the method described by Ghose (1987) at temperatures described in this paper. Activity is expressed such that one unit of enzyme results in the release 1 _mole of glucose-reducing sugar

equivalents from CMC in one minute at the speciWed temperature. Immunodetection of the E1 protein by western analysis was as previously described (Nieves et al., 1995), with the modiWcation of using the ECL western blotting kit (Amersham Biosciences, Piscataway, NJ). Crude protein extracts from the duckweed line were separated by Tris/Glycine SDS-PAGE, followed by transfer to PVDF membrane (Bio-Rad, Hercules, CA). The primary antibody and the secondary antibody were mouse monoclonal antibody against catalytic domain of E1 (E1Cat, original concentration

2.07mg/ml, 1:12,000 dilution) and anti-mouse IgG conjugated with horseradish peroxidase (1:10,000), respectively. The 42,000Da puriWed E1-Cat used as a control was obtained by treating the 72,000Da recombinant E1 holoenzyme expressed in Streptomyces lividans (Nieves et al., 1995; Thomas et al., 1995) with papain (Sakon et al., 1996). The amounts of E1 protein in duckweed extracts were estimated from the intensities of cross-reacting bands using Molecular Dynamics Personal Densitometer SI and Molecular Dynamics Image Quant software (Molecular Dynamics,

Sunnyvale, CA). PuriWed E1-Cat protein (5, 25, 50 ng) was used as the standard to determine the amount of E1 protein. The molecular weight of E1 protein in the transgenic duckweed was estimated using the broad range SDSPAGE molecular weight standards (Bio-Rad, Hercules, CA). The eVects of pH and temperature on E1 activity were tested in a triplicate in order to determine if the properties of the recombinant enzyme were similar to the native E1 protein. Total soluble protein was extracted from transgenic

duckweed line Cel25IX-15 as previously described with sodium citrate buVer (50mM, pH 4.8). The enzyme activity assay was then carried out at 60, 70, 80, 90, or 95 C with the extracts diluted in four volumes of 100mM phosphatecitrate buVers at pH 4, 5, 6, and 7. The heat stability of the recombinant E1 enzyme was also carried out by heating the protein at 60, 70, 80, or 90 C. Aliquots were taken at 15, 30, 45, 60, 120, 180, 240, 300, and 360min, centrifuged, and analyzed for enzyme activity at 80 C and pH 5. In an attempt to improve the eYciency of E1 protein

recovery from the total plant biomass, we tested several diVerent conditions for solubilizing the protein. Fresh duckweed was ground as before, using three diVerent extraction buVers including sodium citrate (50mM, pH 4.8), sodium acetate (50mM, pH 5), or HEPES (50mM, pH 8). Additionally, duplicate preparations of each of the above samples were incubated in a dry bath at 65 C for 5min prior to centrifugation to remove insoluble debris. All the samples were subsequently analyzed for total soluble protein content, CMC-degrading activity, and total E1 protein.

Materials and Methods Study area and samples collection: Samples of wastewater and Lemna minor (duckweed) were collected from the bio-treatment ponds (Fig.1). The bio-remediation

work was commenced at National Agricultural Research Center (NARC), Islamabad in October 2008. The assignment was to reclaim the sewage water of main NARC office buildings and hostels through bioremediation for irrigation purposes. The used water treatment garden project was started in October 2008 and finalized in February 2009. The total area of bio-treatment pond is 0.3 acre and total storage capacity is 264870 (0.265 million gallons) for irrigation (Table 1). All ponds are aerobic while Pond 6 including wetland and Pond 7 is

fish rearing pond. The water and plant samples were collected in replicates.

Plant sampling and heavy metal analysis: Lemna minor plants were collected to study the pattern of heavy metals and comparison of phytoremediation process among seven bio-treatment ponds. From each pond, 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 and put in oven at 70C for 48 hours. The dried plant samples were grinded with pestle and mortar so that their size was 2mm after grinding. Grinded plant samples (1g) were added in 10mL of double acid HNO3:HClO4 (2:1 v/v) in conical flask (300mL) and placed on hot plate in fume hood at 200C. After 24 hours plant samples were digested and filtered, and volume rose to 100mL. All prepared samples were analyzed for heavy metals (Zn, Pb, Cu, Ni,

Cd, Cr, Mn, Fe) by flame atomic absorption spectrophotometer Model No Perkin Elmer Analyst 700 (Ryan et al., 2001). Wastewater sampling and physiochemical analysis: One and half-liter of water samples were collected from all bio-treatment ponds. For sample collection the bottles were washed with hot water followed by distilled water. During collection bottles were filled, rinsed with the sample water 2-3 times, tightly capped and properly labeled. Physical parameters of collected water samples were studied

immediately, which were collected in replicates from all the 7 bio-treatment ponds. In physiochemical 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. It was done during the sampling of water on the spot (Peavy et al., 1985). Qualitative tests that employ the human sense of taste and smell were used for odour purpose (Peavy et al., 1985). Temperature was measured by using a mercury

thermometer of 0oC to 50oC range and with 0.2oC least thermometer of 0oC to 50oC range and with 0.2oC least count. The temperature of water samples was measured on the spot (Trivedi & Gurdeep, 1992). The pH of water samples was determined in laboratory with pH meter (Inolab pH 720). 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 (Trivedi & Gurdeep 1992; Greenberg et al., 2005). The results are

shown in mg/liter using the following formula: TDS = Final weight of china dishinitial weight of china dish/ mL of water sample used X 1000 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. 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 (Zn, Pb, Ni, Mn and Fe) of each water and plant sample were noted (Perkin Elmer Analyst 700). count. The temperature of water samples was measured on the spot (Trivedi & Gurdeep, 1992). The pH of water

samples was determined in laboratory with pH meter (Inolab pH 720). 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 (Trivedi & Gurdeep 1992; Greenberg et al., 2005). The results are shown in mg/liter using the following formula:

Livestock Research for Notes Citation LRRD Rural to of this Newsletter Development Authors paper 22 (7) 2010

Duckweed based bio-remediation of village ponds: An ecologically and economically viable integrated approach for rural development through aquaculture M D Ansal*, A Dhawan and V I Kaur Department of Aquaculture, College of Fisheries, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana-141004, Punjab, India * ansalmd@gmail.com Summary Village pond is an integral part of rural India which is primarily constructed for harvesting rain water and bathing of domestic livestock.

Through suitable scientific interventions these manmade water resources can be utilized for economic gains as well. But unfortunately, they are used only as dumping sites for disposing of human and animal waste which leads to pollution and Eutrophication due to accumulation of excess nutrients (nitrates and phosphates). Although these ponds hold immense potential for producing high quality food through aquaculture for rural development in developing countries but due to poor management and deteriorating water quality, full aquaculture potential of these resources have not been realized so far. It is therefore, vital to reclaim and manage these water bodies to its optimum productivity status through

some appropriate rural scientific technologies.

friendly

Effective waste water treatments through conventional methods, which rely on heavy aeration, are expensive to install and operate. Hence, there is need to explore some non-conventional methods which are not only economically viable and easy to operate but eco-friendly as well. For remediation of village ponds, the first step is to remove the excess nutrients dumped in it. For this purpose, plant based bio-remediation (phyto-remediation) technology is the most promising option. Any aquatic plant that is capable of recovering or extracting nutrients or pollutants and has a fast growth rate coupled with

high nutritive value is an excellent candidate for bio-remediation of waste waters. Such plants grow very fast utilizing waste water nutrients and also yield cost effective protein rich plant biomass as a by-product. Duckweeds hold immense potential for both nutrient recovery and utilization as fodder or feed for livestock including fish. Wastewaterduckweed-aquaculture is a perfect eco-friendly integrated package for converting the waste water nutrients into high quality fish protein and augmenting rural economy through generating employment opportunities and additional food security.

Key words: fish, non-conventional, nutrient recycling, nutrition, phytoremediation, wastewater management Introduction Village ponds hold immense potential for producing high quality food through aquaculture for rural development in developing countries like India. Productivity of these manmade aquaculture resources is however, far below the actual potential due to their poor management. Punjab is one of the progressive states of India and as per Indian Census 2001, it has 12,278 inhabited villages (Statistical Abstracts of Punjab 2006). Each village has at least one village pond

which is used for harvesting rain water, dumping sewage waste and bathing of livestock. Their importance in total fish production of the state can be assessed from the fact that out of the total 10,023 hectare (ha) area under fish farming, about 66.79% (6,695 ha) belongs to village ponds. But due to poor management and deteriorating water quality, full aquaculture potential of these resources has not been realized so far. At present, only 60% of the total village ponds in the state are being utilized for fish culture. Hence, there is still immense scope of utilizing these village ponds for additional fish production.

Village ponds are visited by the domestic cattle (mostly buffaloes) and also receive domestic waste from the village households (including sewage, kitchen waste and detergents), which not only pollutes the water affecting its productivity but also causes nuisance for the villagers due to foul smell and disease outbreak (Figure 1). Hence, it is vital to reclaim and manage these water bodies to its optimum productivity status through some suitable scientific interventions which are not only economically viable but easy enough to be adopted by the illiterate rural population as well.

Figure 1. Polluted village ponds in Punjab, India Since village ponds are a rich source of nutrients (nitrates, phosphates and potassium), these could be utilized or recycled into some other suitable farming system which is capable of not only remediating the pond water

but also converting the recovered nutrients into a much needed cost effective protein rich biomass as a byproduct (Skillicorn et al 1993, Iqbal 1999). Although in India domestic sewagefed aquaculture is almost a century old technique but large-scale usage of sewage for fish culture began in the 1930s in its most thickly populated city Kolkata in West Bengal. It is a unique and the largest operational system in the world (Edwards 2005) to convert waste into consumable products. In view of waste water management, an urban scenario is different from that of a rural scenario. A well developed urban sanitary system carries the city sewage and

waste water (including industrial effluent) to a common disposal site, whereas in rural areas waste water is disposed off in ponds constructed within the village itself due to poor sanitary system. Hence, the rural population is required to play a more active role in waste water management as compared to urban population. Although wastewater-fed aquaculture occurs in several countries in Asia, where it provides food, employment and income for millions of people, especially the poor, the quality of fish raised on urban wastewater is a matter of concern because of the presence of industrial effluent contaminants. However rural waste water, being more organic based, is a more suitable

water resource for producing consumer safe aquaculture products. Effective waste water treatments through conventional methods, which rely on heavy aeration, are expensive to install and operate. Hence, there is need to develop some non-conventional methods, which are not only inexpensive and easy to install but also easy to operate and maintain. In view of this, Phytoremediation is the most suitable bioremediation method for village ponds (ecologically as well as economically) which combines two novel approaches Pollution Prevention and Re-Use.

Phyto-remediation Phyto-remediation is an eco-friendly bio-remediation process of removing pollutants/nutrients from an environment (soil, sediment, water) by using any green plant based system which is not only an energy saving but also a resource recovering system. Suitable aquatic plants for bioremediation of village ponds Any fast growing aquatic plant of high nutritive value is an excellent candidate for bio-remediation of waste water. Many surface floating aquatic plants like water hyacinth (Eichhornia) and duckweeds (Spirodela, Lemna, Wolffia) are well known for their phyto-remediation

qualities (Wolverton 1981, Debusk and Reddy 1987, Akcin et al 1994, Sinha et al 1994, Vajpayee et al 1995, Chandra et al 1997, Zhu et al 1999, Willett 2005). They grow naturally in the village ponds and other nutrient rich standing waters in tropical and sub-tropical climates and are capable of extracting nutrients from these waters which otherwise go waste and cause Eutrophication. The economic potential of any plant species for waste water treatment not only depends largely on its efficiency to remove nutrients under a wide range of climatic conditions and on its growth rate, but also on the possible application of the harvested plant biomass. Although as compared to duckweeds, Eichhornia has a higher

nutrient uptake capability (Wolverton 1976,Reddy and DeBusk 1985) but no economically attractive application of the generated plant biomass has been identified so far due to difficulties in removal of the huge plant biomass from the system. On dry matter (DM) basis, Eichhornia has a fairly good amount of protein content (10-25%) but its high fiber content (17-20%) further reduces its potential for being utilized as animal fodder or feed (Iqbal 1999). In contrast duckweeds, being tiny surface floating plants, are easy to harvest and have a higher protein (15-45% DM basis) and a lower fiber (7-14% DM basis) content. Duckweeds hold an immense potential for both nutrient recovery from village ponds and utilization as

animal fodder or feed due to its fast growth rate, efficient nutrient extracting capability, easy harvesting, high nutritive value and good digestibility (Leng 1999). In USA, duckweed-covered lagoon for tertiary treatment of waste water has been classified as an innovative/alternative technology by the U.S. Environmental Protection Agency (EPA), having great application in rural development in developing countries (Iqbal 1999). Duckweeds Duckweeds are small green plants belonging to family lemnaceae and they grow densely on the water surface forming a mat like cover. Taxonomically they belong to monocotyledons and have four

generas-Lemna, Spirodela, Wolffia and Wolffiella (Figure 2).

Spirodela

Lemna

Wolffia

Wolffiella

Figure 2. Different type of duckweeds About 40 species are reported worldwide (Les et al 2002). Biomass of duckweeds gets doubled in 2-3 days (Iqbal 1999, Sillikorn et al 1993) under ideal conditions of nutrient availability, sunlight, pH (6.5-7.5) and temperature (200C-300C) and can be cultured, harvested and sundried

without much expertise.

cost,

labor

and by

Nutrient uptake/recovery duckweeds

Due to their ability to propagate rapidly by consuming dissolved nutrients from water, duckweeds act as an excellent Nutrient Sink for harvesting nutrients over a short period of time and thus serve as a Nutrient Pump in waste water treatment absorbing various nutrients like nitrates, phosphates, calcium, sodium, potassium, magnesium, carbon, and chloride from the waste water. These nutrients are permanently removed from the system when the plants are harvested.

Besides nutrient extraction, duckweeds has been found to reduce total suspended solids (TSS), biological oxygen demand (BOD) and chemical oxygen demand (COD) in waste water significantly. Korner and Vermaat (1998) reported that depending on the initial concentration of nutrients, duckweed covered systems can remove nitrates (N) and phosphates (P) at rates of 120- 590 mg N/m2/day (73-97% of initial concentration) and 14-74 mg P/m2/day (63-99% of the initial concentration) in three days. Removal efficiencies of 96% and 99% by duckweeds have been reported for BOD and ammonia (NH3), respectively (Alaerts et al 1996).

Reddy and DeBusk (1985) recorded N and P uptake rate of 0.15 g/m2/day and 0.03 g/m2/day by Spirodela polyrrhiza in Florida. whereas, Alaerts et al (1996) found it to be 0.26 g/m2/day and 0.05 g/m2/day, respectively in Bangladesh. Cheng et al (2002) reported maximum N and P uptake of 0.955 mg N/liter/hr and 0.129 mg P/liter/hr by Spirodela punctata. Fat duckweed Lemna gibba have also been found to reduce TSS, BOD, COD, N, NH3, P, phytoplankton crop and fecal coliform counts in waste water by 96.3%, 90.6%, 89.0%, 100%, 82.0%, 64.4%, 94.8% and 99.8%, respectively in 8 days (El-Kheir et al 2007). Nutritive value of duckweeds

The protein content of duckweeds is one of the highest (up to 45%, on DM basis) in the plant kingdom (Fasakin 1999) and has a better array of essential amino acids than most plant proteins and more closely resembles animal protein (Hillman and Culley 1978). Further, its amino acid spectrum especially with regard to lysine (7.5% of total protein) and methionine (2.6% of total protein) is much higher as compared to other plant feed stuffs (Rusoff et al 1980 and Mishra 2007). Duckweeds are highly variable in their composition and it depends on the nutrient status of the water on which they grow (Table 1). They grow slowly on nutrient poor waters and under such

growth conditions have low protein content associated with high fiber, ash and carbohydrate content. In contrast, they grow rapidly on nutrient rich waters and have a high protein content associated with high ash and low fiber content. Table 1. Nutritive value of Duckweeds on DM basis Crud Cru Cru e de de Ash Type Source Protei fat, Fibe , % n, % % r, % Duckw Wolvert eed on and 37.0 3.40 15.6 12.5 (mixed Mcdona / ld 1979 species 6.8- 1.8- 5.7- 12.0 Landolt not 45.0 9.2 16.3 - and

Kandel 27.6 er 1987 Mbagw 12.0 35.05.0u and -* 45.0 15.0 Adeniji 18.0 1988 45.0* Leng et 4.0 9.0 14.0 al 1995 mention * ed by 25.08.0Leng et the 35.0* 4.4 15.0 10.0 al 1995 author) ** Tavares 38.8 3.8 13.2 16.0 et al 2008 Spirod Sutton ela and 29.6 -* -* -* polyrr Ornes hiza 1975 30.52 1.97 17.0 9.45Ansal

20.9 4.1 13.2 20.4 3.8 15.7 Lemna 10.3 28.48 4.75 minor 5 18.38 2.32 -* 28.0 5.0 10.0 Lemna 38.6 9.8 18.7

and Dhawa n 2007 Tacon 13.6 1987 Banerje e and 17.2 Matai 1990 Ahamm -* ad et al 2003 Yilmaz 23.7 et al 2004 Kalita 25.0 et al 2008 19.0Men et

al 1995 Pedraza spp. 36.0 4.5 10.7 8.46 et al 1996 *not reported **Grown on nutrient rich medium *** harvested from a natural lagoon Protein content of duckweeds growing on nutrient poor and nutrient rich waters varies between 15-25% and 35 - 45% (DM basis), respectively (Mbagwu and Adeniji 1988). Root length is a useful indicator of whether pond conditions are appropriate (with respect to nutrients) for production of high protein duckweeds or not (Rodriguez and Preston 1996, Le Ha Chau 1998).

Roots less than 10 mm in length indicate higher protein content in duckweeds than roots more than 10 mm in length and the reverse is true for the fiber content which can be observed very easily under field conditions. Duckweeds are also a rich source of carbohydrates (30-35%), vitamin-A and pigments, particularly beta-carotene and xanthophylls. They contain 92-94% of moisture and harvested biomass can be easily sundried within a period of 24-48 hrs during the dry summer months and 46 days during winter. Sundried as well as pelleted forms of duckweed have been observed in storage for 13 years without any sign of fungal growth and nutrient loss

(Mbagwu 2001) and it has been attributed to the presence of a wax coat on the upper surface of plants which acts as a barrier for fungal growth. A recent finding by Effiong and Sanni (2009) of decreased mold infestation in duckweed (Lemna pausciscostata) incorporated pelleted fish feeds also highlights its value addition potential with great application in feed storage. With 30-40% of protein content (DM basis) nutritive value of duckweeds is comparable to that of soybean. With an annual duckweed yield of 20 t dry weight/ha/yr and protein content of 35% (DM basis), protein productivity of 7 t/ha/yr can be achieved which indicates that relative annual protein

production per unit area through duckweeds is about 10 times higher than that of soybean (Skillicorn et al 1993, Khateeb 2004). Nutritionally also duckweeds have been found to substitute soya and fish meal in feeds of farmed animal like chickens, goats, pigs, ducks and fish (Hillman and Culley 1978, Culley et al 1981, Edwards 1990, Leng et al 1995, Men et al 1995, Anh and Preston 1997, Leng 1999, Iqbal 1999, Landesman et al 2002). Duckweed based rural model for bioremediation The focus on duckweed as a key step in waste recycling is due to the fact it forms the central unit of the recycling

engine which is driven by photosynthesis making it energy efficient, cost effective and ecofriendly. Hence, duckweed based rural bio-remediation model is an effective, cheap and simple way of reclaiming polluted village ponds. For this, divide the village pond into two ponds i.e., duckweed culture pond and fish culture pond, by erecting earthen partition. Only the duckweed pond receives the village waste and duckweed is cultured in surface floating frames (made up of either PVC pipes or split bamboo sticks) to mitigate wind action which can disturb the growing duckweed mat and carry it in the direction of wind (Figures 3 and 4).

Model - I

Model - II

Figure 3. Rural Bio-remediation models for village ponds

Figure 4. Duckweed culture in surface floating bamboo frames (Source: Iqbal 1999)

Remediated water from duckweed pond is released periodically into the fish culture pond. In case it is not possible to divide the village ponds into two parts as desired, it is suggested to culture duckweed in enclosed pens (constructed by erecting partitions made up of bamboo poles and fine mesh net) near the periphery of the ponds (Figures 3 and 5).

Figure 5.

Duckweed culture in

partitioned pens in village pond (Source: Leng 1999) In Punjab, a pilot duckweed project for bio- remediation of village pond was initiated in 2001 by the State Government in collaboration with Punjab State Council for Science and Technology (PSCST) in villages Sanghol and Chanarthal kalan in District Fatehgarh Sahib (Singh et al 2003). Under this project, village ponds were divided into a duckweed pond and a fish culture pond. Bioremediated water from the duckweed pond was used for poly-culture of carps (Indian major carps and exotic carps) in the fish culture pond and harvested duckweed biomass was

utilized to feed the fish. Encouraging results in terms of enhanced fish production from the bio-remediated village pond has lead to continuation of the project till date. For the first couple of years the project was operated and maintained by Sulabh International (an Indian based social service organization which works to promote human rights, environmental sanitation, non-conventional sources of energy, waste management and social reforms through education), but now this project is operated and maintained by the Gram Panchayat (elected village administration) of the villages itself. After the success story of first duckweed pilot project in Punjab, another project was taken up by PSCST in village Sandhua in

district Ropar in 2003 and many other are in pipeline (PSCST 2005). Duckweed harvesting schedule Regular harvesting of duckweed helps in regular extraction or recovery of nutrients from the village ponds (Figures 6-7).

Figure 6. Harvesting Figure 7. Harvested of duckweed duckweed

A well planned harvesting schedule is required to maintain vigorous growth of duckweed and nutrient removal. It should be designed according to the growth rate of duckweeds, usually having biomass doubling times ranging from 2 to 3 days. Hence, removal of half of the duckweed biomass or cover on every third day is a practical option which not only ensures development of full duckweed cover over the pond surface within a short period but also helps in blocking the sunlight from entering into the waste water. This is required for preventing growth of unicellular and filamentous algae in the waste water, which otherwise grow very fast and compete for nutrients affecting growth and quality of duckweed.

Duckweed productivity from 10 to 50 t (dry biomass)/ha/yr has been reported (Gijzen and Khondker 1997) from different parts of the world. Fresh duckweed yields in the range of 0.5 to 1.5 t/ha/day have been reported in Bangladesh which corresponds to production of 185 to 550 t of fresh or 13 to 38 t of dry duckweed biomass/ha/yr (Skillicorn et al 1993). Utilization of duckweed biomass Huge biomass of duckweed harvested from the village ponds can be utilized in both fresh and dried form (Figures 8-9) by the destitute rural population for economic gains through one of the following options.

Figure 8. Feeding fresh Figure 9. Sunduckweed to fish dried duckweed (Source: Iqbal 1999) Duckweeds as fish feed Prior to 1988, duckweeds had been used only in commercial applications to treat wastewater in North America. In 1989 staff of a non-governmental organization based in Columbia, Maryland, The PRISM Group initiated a pilot project in Bangladesh to develop farming systems for

duckweed and to test its value as a feed for herbivorous/omnivorous fishes like carps and tilapia. The results of the pilot operations were extremely promising and dried duckweed meal provided an excellent substitute for expensive conventional feed ingredients like soybean and fish meal (Iqbal 1999). Fresh duckweed is converted efficiently to live weight by fish. Feed conversion ratio of 1.2 to 3.3 for Spirodela in carps and 1.6 to 3.3 for Lemna in tilapia has been recorded by Gijzen and Khondken (1997). Duckweed incorporated dry diets have also been found to support growth in not only herbivorous or omnivorous fishes like carps and tilapia but in high protein demanding

carnivorous fishes like catfishes and snakeheads as well (Table 2). Table 2. Summary of positive reports on inclusion of dried duckweeds in fish feed Inclusio Refere Type Fish n level nce 30% fish Fasakin meal Oreochromi et al replacem s niloticus 2001 ent Spirod Ansal ela Labeo and polyrr rohita, Dhawa hiza Cirrhinus 20 % n 2007, mrigala Ansal Cyprinus et al carpio 2008

Lemna minor 40%

Cyprinus carpio

Channa 50% striatus Cyprinus carpio, 100% Catla catla, Azim duckwe Barbodes and ed gonionotous Wahab feeding *, 2003 Oreochromi s niloticus Yilmaz Cyprinus 20% et al carpio 2004 13.2% Labeo Guru rohita and Patra

Devaraj et al 1981 Raj et al 2001

20% 40% Lemna polyrr 30% hiza Wolffi 10% a spp. Duckw eed 20 % (Mixe d / 60% spp. not mentio ned by

2007 Labeo Das et rohita al 2007 Tavares Oreochromi et al s niloticus 2008 Bairagi Labeo et al rohita 2002 Ctenophryn Verma godon idella 1989 Robinet Ictalurus te et al punctatus 1980 Indian majorIqbal carps, 1999 Chinese carps , Tilapia

Oreochromi 100% s niloticus, duckwe Cyprinus Thy et ed carpio, al 2008 the authors) feeding Cirrhinus mrigala *Puntius gonionotous Carps In India carp poly-culture system contributes more than 80% of the total aquaculture production where Indian major carps (Catla catla, Labeo rohita and Cirrhinus mrigala) and exotic carps (Cyprinus carpio, Ctenopharyngodon idella and Hypophthalmichthys molitrix) are

cultured together. Among these species grass carp (Ctenophryngodon idella) is the primary consumer of aquatic plants (herbivorous) including duckweeds. Catla (Catla catla) and common carp (Cyprinus carpio) also compete aggressively for available duckweed feed and consume it directly. In Bangladesh, about 10t/ha/yr fish productivity has been reported from duckweed fed carp poly-culture ponds (Iqbal 1999). Another exotic carp, Thai silver barb- Barbodes gonionotous (Puntius gonionotous) has also been reported to grow fast in duckweed fed polyculture system (Azim and Wahab 2003).

Incorporation of dried duckweed, Lemna minor at 40 % in the supplementary diet of common carp, Cyprinus carpio (Devaraj et al 1981) revealed higher specific growth rate besides lowering the feed cost significantly. In Bangladesh, higher fish yields have been recorded in a poly-culture system, comprising Indian major carps, Chinese carps and tilapia, when fed with diets containing 60% sewage-grown mixed duckweeds and 40% mustard oil cake (Iqbal 1999). Guru and Patra (2007) also reported higher specific growth rate in Labeo rohita fingerlings fed with diets having 13.2% dried Lemna powder. Das et al (2007) recorded 205% higher weight gain and about 105% higher food

conversion in Labeo rohita fed with diets containing 20% dried Lemna minor powder and also saved about 20% of feed cost. Central Inland Fisheries Research Institute (CIFRI), India reported increased growth rate in grass carp when fed with diets containing 10% Wolffia (Verma 1989). Studies conducted at Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Punjab, India also revealed significantly higher weight gain in carps like Labeo rohita (20.60%), Cirrhinus mrigala (26.80%) and Cyprinus carpio (70%) fed with diets containing 20% sundried Spirodela and saved up to 50% on feed cost (Ansal and Dhawan

2007, Ansal et al 2008) by 100% replacement of animal protein supplement in the traditional diets. Tilapia Nile Tilapia, Oreochromis niloticus, being extremely flexible in its feeding habits, readily consumes Lemna and Wolffia species along with phytoplankton and detritus. Skillicorn et al (1993) reported that when fresh duckweed was used as a single nutritional input for tilapia in earthen ponds, fish production reached 7.5 t/ha/yr in Bangladesh. Fasakin et al (2001) developed a low cost feed for tilapia, Oreochromis niloticus L. by utilizing solar-dried duckweed

(Spirodela polyrrhiza) up to 30% dietary inclusion as a replacement for fishmeal in practical diets. Dry duckweed has also been reported to replace up to 50% of the commercial tilapia feed without adverse effects on fish performance (Essa 1997, Tavares et al 2008). Catfishes Robinette et al (1980) obtained weight gain and food conversion equal to that of the standard feed in catfish (Ictalurus punctatus) fed with feed containing 20% dry duckweed. Effiong et al (2009) also reported 10% fish meal replacement with duckweed meal (Lemna pauciscostata) in African

catfish- Heterobranchus longifilis diet. Snakeheads Incorporation of duckweed, Lemna minor at 50% in the supplementary diet of snakehead, Channa striatus also resulted in higher specific growth rate and weight gain besides lowering the feed cost significantly(Raj et al 2001). Other species In Australia, Jade Perch (Scortum barcoo) has been reported to actively consume and gain weight (with 100% survival) solely on fresh duckweed harvested from effluent treatment

plant (Willett et al 2003). Presence of good amount of carotenoids and pigments in duckweed can stimulate crustacean growth (Landesman et al 2002). Fletcher and Warburton (1997) have found that decomposed Spirodela is as effective as commercial pelleted feed for culturing redclaw crayfish, Cerax quadricarinatus. Vermicomposting of duckweeds As fresh duckweed is characterized by high amounts of nitrogen and phosphorus, compost made from duckweeds is also expected to be rich in these macronutrients. Kostecka and Kaniuczak (2008) developed a high quality macronutrient rich (N, P & K)

vermicompost from duckweed (Lemna spp.) biomass by using Eisenia fetida (SAV.) earthworms. Hence, vermicomposting of harvested duckweed biomass further corroborates its potential for utilization in environmental reclamation including aquaculture as well as agriculture. Production of value added products from duckweeds Besides quality protein resource, duckweeds are also a good resource of starch. Hence, there is great scope of production of value added products like protein concentrate and ethanol from duckweeds. About 64.4 % crude protein content has been reported in

leaf protein concentrates prepared from Spirodela polyrrhiza (Fasakin 1999), which can be used as feed supplement not only in animal feeds but also for human consumption. Spirodela polyrrhiza grown on anaerobically treated swine waste water has been found to have a starch content of 45.8% on DM basis and its enzymatic hydrolysis yielded a hydrolysate with a reducing sugar content corresponding to 50.9% of the original duckweed biomass. Further, fermentation of the sugar hydrosylate by yeast gave an ethanol yield of 25.8% of the original dry duckweed biomass which reflects an additional scope of harvested duckweed biomass

in ethanol production (Cheng and Stomp 2009). Suggestions Most of the village ponds are constructed without proper planning (location and layout) and drainage system. Ponds located in the middle of the inhabited area of the village are difficult to drain out and have little scope of alterations required for developing duckweed based bioremediation models. However ponds at the outskirts of the village are easy to manage as its nutrient rich water can be utilized for irrigating the adjoining agricultural fields. Hence, for full scale commercial utilization of duckweed based bio-remediation

models, construction of village ponds are required to be well planned with respect to location, layout and drainage facility. State government also needs to promote duckweed based rural models for bioremediating village ponds through educating the people and introducing some rural welfare schemes integrated with village ponds for employment and income generation. Variety of animals like cows, buffaloes, goats, sheep, pigs, chicken and fish are being reared in rural India for milk, meat, wool and eggs. Utilization of duckweeds as fodder or feed ingredient for these animals is also required to be popularized among the rural population. Increased local demand will certainly promote

duckweed ponds.

aquaculture

in

village

Conclusions

Although polluted, village ponds are rich source of nutrients like nitrate and phosphate which can be recovered by phyto-remedition. It is an affordable technology utilizing plants as environmental cleansers in wastewater management. On one hand manure and fertilizers are getting costlier day by day and on the other hand we have resources like village ponds where the much needed nutrients are lying free of cost. Therefore, recovering this valuable nutrient resource and recycling

into some productive system makes sense both ecologically and economically. Hence, an eco-friendly approach of duckweed culture in nutrient rich village ponds will not only help in free of cost extraction of nutrients (which otherwise pollute the water and go waste) in the form of protein rich duckweed but also bio-remediate the village ponds and make them a more suitable water resource for aquaculture. Wastewater-duckweed-carp polyculture makes the perfect integrated package for pollution control and re-use of recovered nutrients. Bio-remediation will not

only augment contribution of village ponds to total aquaculture production of any developing countries like India but also generate employment opportunities and additional food security for its rural population. Moreover in view of increased pressure on land over the years for production of food and fodder (due to ever increasing population, urbanization, industrialization etc.), utilization of an alternate resource for the purpose makes sense. In this direction, village ponds hold ample scope not only for high quality food production through aquaculture but also releasing

pressure on the underground water resources. Although proven but full scale commercial application of duckweed based rural models for bio-remediation of village ponds has not achieved a major breakthrough so far. It is possible only through educating the rural population regarding its economic and environmental benefits and by providing them the required technical guidance and financial assistance.

References

Ahammad M U, Swapon M S R, Yeasmin T, Rahman M S and Ali M S 2003 Replacement of sesame oil cake by duckweed (Lemna minor) in broiler diet. Pakistan Journal of Biological Science 6 (16): 14501453.http://scialert.net/qredirect.php? doi=pjbs.2003.1450.1453&linkid=pdf Akcin G, Saltabas O and Afsar H 1994 Removal of lead by water hyacinth (Eichhornia crassipes). Journal of Environment Science and Health 29 (10): 2177-2184. Alaerts G J, Rehman M and Kelderman P 1996 Performance analysis of a full-scale duckweed covered lagoon. Water Research 30: 843-852.

Anh N D and Preston T R 1997 Evaluation of protein quality in duckweed (Lemna spp.) using a duckling growth assay. Livestock Research for Rural Development 9(2): http://www.lrrd.org/lrrd9/2/anh9 2.htm Ansal M D and Dhawan A 2007 Spirodela for low cost carp feed Formulation. Abstract In: 8th Asian Fisheries Forum, Kochi, India, November, 20-23, 2007, p 164 Ansal M D, Dhawan A and Kaur V I 2008 Efficacy of duckweedSpirodela in carp diet. Indian Journal of Ecology 35 (2): 139-142.

Azim M E and Wahab M A 2003 Development of a duckweed fed carp polyculture system in Bangladesh. Aquaculture 218 (1-4): 425-438. Bairagi A, Sarkarghosh K, Sen S K and Ray A K 2002 Duckweed (Lemna polyrrhiza) leaf meal as source of feedstuff in formulated diets for rohu (Labeo rohita Ham) fingerlings after formulation with a fish intestine bacterium. Bioresource Technology 85 (1): 17-23. Banerjee A and Matai S 1990 Composition of Indian Aquatic plants in relation to utilization as animal forage. Journal of Aquatic Plant Management 28: 69-73.

Chandra P, Sinha S and Rai U N 1997 Bioremediation of chromium from water and soil by vascular aquatic plants. In: Kruger E L, Anderson T A and Coats J R (Editors), Phytoremediation of Soil and Water Contaminants. Washington, D.C., American Chemical Society. PP. 274-282. Cheng J J and Stomp A M 2009 Growing duckweed to recover nutrients from wastewater and for production of fuel ethanol and animal feed. Clean Soil, Air, Water 37(1): 17-26. Cheng J, Ben A, Bergmann B A Classen J J, Stomp A M and

Howard J W 2002 Nutrient recovery from swine lagoon water by Spirodela punctata. Bioresource Technology 81 (1): 81-85. Culley D D, Rejmankova E, Kvet J and Frye J B 1981 Production, chemical quality, and use of duckweeds (Lemnaceae) in aquaculture, waste management and animal feed. Journal of World Mariculture Society. 12: 27-49. Das N, Sharma B K, Sharma L L, Saini V P and Upadhyay B 2007 Efficacy of certain formulated diets of plants and animal origin In relation to growth of rohu (Labeo rohita) fry. Fishing Chimes 27(7): 2426.

Debusk T A and Reddy K R 1987 BOD removal in floating aquatic macrophyte-based waste water treatment systems. Water and Science Technology 19: 273-279 Devaraj K V, Krishna Rao D S and Keshavappa G Y 1981 Utilization of duckweed and waste cabbage leaves in the formulation of fish feed. Journal of Agriculture and Science 15: 132-135. Edwards P 1990 An alternative excreta re-use strategy for aquaculture: the production of high protein animal feed. In: Edwards P, Pullin R S V (Editors). Proc. International Seminar on waste water

reclamation and re use for aquaculture, Calcutta, India, December 1988, pp. 209-221. Edwards P 2005 Development status of, and prospectus for, wastewater-fed aquaculture in urban environment. In: Costa-Pierce B, Desbinnet A, Edwards P and Baker D (Editors). Urban Aquaculture. CABI Publishing,UK. pp. 45-60. Edwards P, Polprasert C and Wee K L 1987 Resource recovery and health aspects of sanitation. AIT Research Report No. 205. pp. 324. Effiong B N and Sanni A 2009 Effect of duckweed meal on the rate of mold infestation in stored

pelleted fish feed. Journal of American Science 5 (1): 2934 http://www.sciencepub.net/report/0 102/03_0537_mold_infestation_feeds -Effiong.pdf Effiong B N, Sanni A and Fakunle J O 2009 Effect of partial replacement of fish meal with duckweed (Lemna pauciscostata) meal on the growth performance of Heterobranchus longifilis fingerlings. Report and Opinion 1 (3): 7681http://www.sciencepub.net/report/0 103/14_0816_FINGERLINGS_pub_r eport0103.pdf El-Kheir W A, Ismail G, El-Nour A, Tawfik T and Hammad D 2007 Assessment of the efficiency of

duckweed (Lemna gibba) in wastewater treatment. International Journal of Agriculture and Biology 5: 681-689. Essa M A 1997 Utilization of some aquatic plants in diets of Nile tilapia, Oreochromis niloticus, fingerlings. Egyptian Journal of Aquatic Biology and Fisheries 1 (2): 19-34. Fasakin E A 1999 Nutrient quality of leaf protein concentrates produced from water fern (Azolla africana) and duckweed (Spirodella polyrrhiza L.Schleiden). Bioresource Technology 69 (2): 185-187.

Fasakin E A, Balogun A M and Fasuru B E 2001 Use of duckweed, Spirodella polyrrhiza L. Schleiden, as a protein feed stuff in practical diets for tilapia, Oreochromis niloticus L. Aquaculture Research 30 (5): 313318. Fletcher A and Warburton K 1997 Consumption of fresh and decomposed duckweed Spirodela sp. by Redclaw crayfish, Cerax quadricarinatus. Aquaculture Research 28: 379-382. Gijzen H J and Khondker M 1997 An overview physiology, cultivation and applications of duckweed. Report. Annex I.

Literature Review. Duckweed (DWRP), Dhaka, Bangladesh. 53pp. Guru S R and Patra A K 2007 Evaluation of nutritional impact of water weed-based feeds on the growth of Labeo rohita. Fishing Chimes 27 (4):28-30, 35-36. Hillman W S and Culley D D 1978 The uses of duckweed. American Scientist 66: 442-451. Iqbal S 1999 Duckweed Aquaculture: Potentials, Possibilities and Limitations for combined Waste water Treatment and Animal Feed Production in Developing Countries. SUNDEC Report no. 6/99.Switzerland.

Kalita P, Mukhopadhyay P K and Mukherjee A K 2008 Supplementation of four nonconventional aquatic weeds to the basal diet of Catla catla and Cirrhinus mrigala fingerlings: Effect on growth, protein utilization and body composition of fish. Acta Ichthyologica Et Piscatoria 38 (1): 2127. Khateeb N A 2004 Duckweed use for sewage treatment and fodder production in Palestine. In: Water for life in the middle east 2nd IsraeliPalestinian International Conference, Turkey 10-14 October

2004. http://www.ipcri.org/watconf/p apers/nader.pdf Korner S and Vermaat J E 1998 The relative importance of Lemna gibba L., bacteria and algae for the nitrogen and phosphorus removal in duckweed-covered domestic waste water. Water Research 32: 3651-3661. Kostecka J and Kaniuczak J 2008 Vermicomposting of duckweed (Lemna minor L.) biomass by Eisenia fetida (Sav.) earthworm. Journal of Elementology 13 (4): 571-579. Landesman L, Yamamoto Y and Goodwin J 2002 Nutrition value of waste water grown duckweed for fish

and shrimp feed. World Aquaculture 12: 39-40. Landolt E and Kandeler R 1987 Biosystematics investigations in the family of Duckweeds (Lamnaceae). Veroff. Geobot. Institute. ETH. Zurich. Volume 2: pp 42-43. Le Ha Chau 1998 Biodigester effluent verses manure, from pigs or cattle, as fertilizer for duckweed (Lemna spp.). Livestock Research for Rural Development 10 (3): 16. http://www.lrrd.org/lrrd10/3/chau2. htm Leng R A 1999 Duckweed: A tiny aquatic plant with enormous potential

for agriculture and environment. Animal Production and Health Division, University of Tropical Agriculture Foundation, Phnom Penh (Combodia). FAO Rome (Italy). 108 p. http://www.fao.org/ag/AGAinfo/res ources/documents/DW/Dw2.htm Leng R A, Stambolie J H and Bell R E 1995 Duckweed- A potential high protein feed resource for domestic animals and fish. Livestock Research for Rural Development. (7) 1: http://www.lrrd.org/lrrd7/1/3.htm Les D H, Crawford D J, Landolt E, Gabel J D and Kimball 2002 Phylogeny and systematics of Lemnaceae, the duckweed family. Systematic Botany 27: 221-240.

Mbagwu I G 2001 The effect of long term storage on the nutrient characteristics of Duckweed (Lemna pauciscostata Hegelm). Journal of Arid Agriculture 11: 147-149. Mbagwu I G and Adeniji H A 1988 The nutritional content of duckweed (Lemna pauciscostata Hegelm) in the kainji lake area, Nigeria. Aquatic Botany 29: 357-366. Men B X, Ogle B and Preston T R 1995 Use of duckweed (Lemna spp) as replacement for soya bean meal in basal diet of broken rice for fattening ducks. Livestock Research for Rural

Development (7) 3: http://www.lrrd.org/lrrd7/3/2.htm Mishra B 2007 On Duckweed, the multi-use aquatic plant. Fishing Chimes 27 (5): 11-13. Pedraza G, Conde N and Chara J 1996 Evaluacion de un sistema de descontaminacion de agues a traves de la producccion de organismos y plantas acuaticas. Report CIPAV, Cali. 106 pp. PSCST 2005 Duckweed technology for rural waste water treatment. Punjab State Council for Science and Technology. Chandigarh, Punjab, India. www.punenvis.nic.in/water/tec hnologies10.htm

Raj A J A, Muruganandam, M, Marimuthu, K and Haniffa M A 2001. Influence of aquatic weed (Lemna minor) on growth and survival of the fingerlings of Channa striatus. Journal of the Inland Fisheries Society of India 33 (1): 5964. Reddy K R and Debusk W F 1985 Nutrient removal potential of selected aquatic macrophytes. Journal of Environmental Quality 14 (4): 459462. Robinette H R, Brunson H R and Day E J 1980 Use of duckweed in diets of channel cat fish. Mississippi Agricultural Experiment

Station. Publication No. Mississippi State 13pp.

4532.

Rodriguez L and Preston T R 1996 Use of effluent from low cost plastic biodigesters as fertilizer for duckweed ponds. Livestock Research for Rural Development. 8 (2): 7281. www.lrrd.org/lrrd8/2/lylian2.htm Rusoff L L, Blakeney EW and Culley D D 1980 Duckweeds (Lemnaceae): A potential source of protein and amino acids. Journal of Agriculture Food and Chemistry 28: 848-850. Singh K, Singh B and Walia S S 2003 Fategarh Sahib tops in introducing duckweed technology at

village Chanarthal Kalan and Sanghal. Punjab Fisheries Bulletin. XXIII, 3134. Sinha S, Gupta M and Chandra P 1994 Bioaccumulation and toxicity of Cu and Cd in Vallisnaria spiralis (L). Environment Monitoring and Assessment 33 (1): 75-84. Skillicorn P, Spira W and Journey W 1993 Duckweed Aquaculture- A new aquatic farming systems for developing countries. The World Bank. 76 pp. Washington DC. http://www.p2pays.org/ref/09/08 875.htm

Statistical Abstracts of Punjab 2006 www.punjabgovt.nic.in/punjabat aglance/some_figures.htm Sutton D L and Ornes W H 1975 Phosphorous removal from static sewage effluent using duckweeds. Journal of Environmental Quality. 4: 367-370. Tacon, A G J 1987 Nutrient sources and composition. In: The nutrition and feeding of farmed fish and shrimp-A training manual. FAO, Brasilia, Brazil. 137 pp. http://www.fao.org/docrep/field/0 03/ab470e/AB470E00.htm Tavares F de A, Roudrigues, J S R, Fracalossi D M, Esquivel J and

Roubach R 2008 Dried duckweed and commercial feed promote adequate growth performance of tilapia fingerlings. Biotemas 21 (3): 91-97. http://www.biotemas.ufsc.br/v olumes/pdf/volume213/p91a97.pdf Thy S, Borin K, Vanvuth T, Buntha P and Preston T R 2008 Effect of water spinach and duckweed on fish growth performance in poly-culture ponds. Livestock Research for Rural Development. 20 (1): 111.http://www.lrrd.org/lrrd20/1/sant2 0016.htm Vajpayee P, Rai U Tripathi R D and 1995 Bioremediation effluent by aquatic N, Sinha S, Chandra P of tannery macrophytes.

Bulletin of Environmental Contamination and Toxicology 55 (4): 546-553. Verma J P 1989 Utilization of aquatic weeds as fish feed. In: Compendium of Lectures (Vol.2) of Training Programme on Integrated Fish Farming for Tripura Fisheries Officers. CIFA (ICAR), Bhubaneshwar, 12-31, January 1989. pp. 42-45. Willett D 2005 Duckweed-based wastewater treatment systems: Design aspects and integrated reuse options for Queensland conditions. Queensland Department of Primary Industries and Fisheries. DPI & F Publications

Brisbane. 24 pp.http://www.dcm.nt.gov.au/__data/a ssets/file/0015/43143/14e.pdf Willett D, Rutherford B, Morrison C and Knibb W 2003 Tertiary treatment of Ayr municipal wastewater using bioremediation: a pilot study. Report to the Burdekin Shire Council and the Burdekin Rangelands Reef Initiative. Queensland Department of Primary Industries. 14 pp. Wolverton B C 1976 Making aquatic weeds useful: some perspectives for developing countries. Report of panel on the Advisory Committee on Technology Innovation, Board of Sciences and Technology for

International Development Commission on International Relations, National Academy of Sciences, Washington, D.C., USA. http://books.google.fr/books? id=n2krAAAAYAAJ&printsec=front cover&dq=Making+aquatic+weeds+u seful: +some+perspectives+for+developing +countries&source=bl&ots=XXAE7y 0C8W&sig=QCxMa2DKlGowsE0fX wZ8390VyyI&hl=fr&ei=YpoTTNzN KpGI4ganwe3XDA&sa=X&oi=book _result&ct=result&resnum=1&ved=0 CBUQ6AEwAA#v=onepage&q&f=fa lse Wolverton B C 1981 Water hyacinth for controlling water pollution. In: Varhney C K (Editor), Water

pollution and management review. South Asian Publishers Pvt. Ltd. Madras. pp. 47-51. Wolverton B C and McDonald R C 1979 Energy from aquatic plant wastewater treatment. In: NASA Technical Memorandum. Wastewater treatment systems (NASA) 18 pp. Yilmaz E, Akurt I and Gunal G 2004 Use of duckweed, Lemna minor, as a protein feedstuff in practical diets for common carp, Cyprinus carpio, fry. Turkish Journal of Fisheries and Aquatic Sciences 4: 105109.http://www.trjfas.org/pdf/issue_4 _2/105_109.pdf

Zhu Y L, Zayed A M, Qian J H, Souza M and Terry N 1999 Phytoremediation of trace elements by wetland plants II. Water hyacinth. Journal of Environmental Quality 28: 339-344.

Received 1 February 2010; Accepted 16 May 2010; Published 1 July 2010 Go to top