CHAPTER 6: NUTRIENT CONCENTRATION AND REPLENISHMENT OF SLURRY
6.3 Materials and methods
conditions and harvest regimes affects biomass production and quality of W. arrhiza. The objective of this study was to assess influence of nutrient concentration and replenishment interval of swine lagoon water (SLW), and harvest frequency on W. arrhiza growth performance and nitrogen uptake.
6.3 Materials and methods
6.3.1 Swine lagoon water sampling and duckweed conditioning
The study was conducted at University of KwaZulu-Natal in Pietermaritzburg (29o 37' 33.9″S; 30o 24' 14″E), South Africa from February to May 2016. Fifty litres of swine lagoon water (SLW), was sampled from lagoons adjacent to pig sties at Baynesfield Estate (29ᵒ45'S and 30ᵒ20'E). The SLW was collected from eight different sites of the lagoons and mixed thoroughly in 25 litre plastic containers. The SLW was screened through a 50 µm screen and stored at 4ºC in a cold room. One- litre samples of SLW were analysed for pH, electrical conductivity (EC), total Kjedhal nitrogen (TKN), total P (TP) and soluble cations by Talbot and Talbot laboratories (Table 6.1). The wastewater had high TKN and higher total P and K than FAO thresholds, while all other properties were within acceptable limits.
About 60 kg wet mass of duckweed species W. arrhiza were randomly collected from a pond that received pig slurry effluent at Ukulinga (29o39'S, 30o24'E) research farm of the University of KwaZulu-Natal. The duckweed was transported to the laboratory as a dense suspension.
Extraneous materials such as insects, grass and small sticks were then removed by passing the suspension through a set of sieves (from 1000 to 50 µm), before rinsing with distilled water.
96 Table 6.1 Elemental composition of swine lagoon water passed through a 0.50 µm screen.
Parameter Mean±se SA Irrigation water
quality a
FAOb EC (dSm-1) 5.94±0.03
pH 7.5 ±0.1 6.5-8.4 6.5-8.4
TKN (mgL-1) 587.5 ±13 - -
TP (mgL-1) 42.3 ±3.0 - <2
K(mgL-1) 381.0 ±2.0 - <2
Ca (mgL-1) 57.5 ±0.5 - <400
Mg (mgL-1) 27.5 ±0.5 - <61
Al (µgL-1) 80.0 ±3.0 <5000 5000
Mn (µgL-1) 75.5 ±0.5 <200 200
Zn (µgL-1) 225 ±9 <1000 2000
Cu (µgL-1) 79.0 ±4 <200 200
Co (µgL-1) 7.8 ±0.2 <50 50
Cr (µgL-1) <1 <100 100
Pb (µgL-1) <1 <200 5000
Cd (µgL-1) <1 <10 10
TKN = Total Kjedhal Nitrogen; TP = Total P; se = standard error; a Department of Water Affairs and Forestry (1996); bFood and Agriculture Organisation (Ayers and Westcot, 1985; Pescod, 1992)
A duckweed sample was oven dried at 60oC to constant weight before elemental analysis.
Characterisation of W. arrhiza showed that it had 32% C, 5.8% N, 0.95% P, 7.4% K, 0.6% Ca and 0.3% Mg. In addition, it had 277 mg Al kg-1 and 9055 mg Fe kg-1, 151 mg Mn kg-1, 128 mg Zn kg-1 and 6.9 mg Cu kg-1.
The SLW (TKN 588 mgL-1) was diluted to 5% using distilled water to ensure that the N content was lower than 60 mg N L-1 to avoid ammonia toxicity before conditioning a 100 g sample of W.
arrhiza in a 25-litre dish for a week at room temperature of 22-25ºC.
97 6.3.2 Effects of replenishment of SLW and harvesting frequency on duckweed
biomass and N uptake
Effect of SLW replenishment and harvest frequency on duckweed biomass was based on the 5%
SLW concentration that yielded the highest duckweed biomass in earlier experiments. A 3 x 3 factorial experiment arranged in a randomised complete block design, with three replicates, was set up in the growth room. The treatments were three levels of SLW replenishment (twice a week, once a week and non-replenishment) and three levels of harvest frequency (twice a week, once a week and once after two weeks). Blocking was against airflow direction in the growth room.
Plastic containers measuring 29 cm x 19cm x10 cm (surface area 545 cm2) were filled to a depth of 5 cm with dilute solution of SLW. The containers were inoculated with 150 gm-2 fresh duckweed for a single layer cover. Al-Nozaily (2001) reported that duckweed growth rate decreased as biomass accumulated to the point that fronds start overlapping each other. The growth room was illuminated with fluorescence lamps at 9 000 lux on a 12 hrs light/dark cycle at 26oC. Solution lost through evaporation was replaced using distilled water. Containers were placed on fixed platforms at a height of a metre from the ground in the growth room. Harvesting was as described in Section 6.3.5, before determination of dry matter, C and N content. The experiment lasted two weeks.
6.3.3 Effects of concentration of SLW and its replenishment on duckweed biomass and N uptake
A 3 x 3 factorial experiment arranged in a randomised complete block design, with three replicates, was set up in the growth room to determine the effects of SLW concentration and time of its
98 replenishment on duckweed biomass, N content and uptake. The SLW was diluted with distilled water to three treatment levels (5, 10 and 15% of the original concentrations) based on results of preliminary studies. Solution replenishment had three levels (twice a week, once a week, and non- replenishment). The diluted solutions were filled to a depth of 4.9 cm in 21cm x 14.5 cm x 6 cm (L x W x D) plastic containers (surface area 411 cm2). The containers were inoculated with 150 gm-2 fresh duckweed for a single layer cover. Harvest, as detailed in Section 6.3.5, was once every week to avoid overcrowding. The experiment lasted two weeks.
6.3.4 Effects of concentration of SLW and harvesting frequency on duckweed biomass and N uptake
Effect of SLW concentration and harvesting frequency on W. arrhiza biomass, N content and uptake was studied by setting up a 2 x 3 factorial experiment arranged in a randomised complete block design, with three replicates in a growth room. The treatments were two levels of SLW concentration (5 and 10% dilutions with distilled water) and three levels of harvesting frequency as in Section 6.3.2. The experiment was conducted without solution replenishment. Plastic containers (surface area 545 cm2) as in Section 6.3.2, were filled to a depth of 5 cm with dilute solution of SLW. The containers were inoculated with 150 gm-2 fresh duckweed. The growth room conditions and other management of the trial were as described in Section 6.3.2. Harvesting and tissue analysis were as described in Section 6.3.5, under harvesting and analyses of duckweed biomass and swine lagoon water. Samples of the solution (5 ml) were drawn and analysed for NH4+, NO3- and PO43- at 3, 7, 10 and 14 days. The experiment lasted 14 days.
99 6.3.5 Harvesting and analyses of duckweed biomass and swine lagoon water
Duckweed biomass was harvested from each container using a strainer and blocker to remove fronds (combination of leaf and stem) from 50% of the surface area. Removal of 50% duckweed allowed coverage of water surface at a rate fast enough to supress algal growth. Water was allowed to drip for five minutes from duckweed, using a strainer, and wet mass was measured after lightly bloating the biomass on a paper towel. Dry weight was measured after oven drying at 60ᵒC to constant weight. The duckweed biomass was passed through a 500 µm screen and analysed for C and N by the LECO Trumac CNS auto-analyser Version 1.1x (LECO Corporation, 2012). The NH4+, NO3- and PO43- in the water sampleswere analysed using the Thermo Scientific Gallery Automated Chemistry Analyser following methods by Rice et al. (2012). Mineral-N was the sum of ammonium and nitrate-N. Average growth rate over the period of culturing was determined as follows: Average growth rate = increased dry weight/ area/ time (gm-2day-1) (Zhao et al., 2014b).
6.3.6 Data analysis
All data were subjected to analysis of variance (ANOVA) using GenStat 14th edition. Least significant differences (LSD) (at p<0.05) were used to separate the treatment means.
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