Effect of sludge resulting from biocoagulation-flocculation process on Abelmoschus esculentus L. growth

The current study was performed to determine the effect of sludge resulting from biocoagulation-flocculation process on okra (Abelmoschus esculentus L.) growth. The effectiveness of the resultant sludge collected after coagulation-flocculation of aquaculture effluent using biocoagulant-flocculant was compared with garden soil (nutrient rich medium) and also with sand (nutrient deficiency medium). The growth of plants in terms of physical observation and also the agronomic parameters (the number of leaves and the height of plants) was monitored for 8 weeks. At the end of the exposure period, there is no significant difference in terms of the number of leaves and also the height of plants between plants in garden soil and plants in pots with the resultant sludge. In contrast, the agronomic parameters for plants in pot with sludge were significantly higher than plants in pot with sand only. As for the relative growth rate (RGR) of plants, plants in pots with sludge were the highest (3.84 g/week) compared to garden soil (3.52 g/week) and sand only (0.25 g/week). It can be concluded that sludge generated from the coagulation-flocculation process of aquaculture effluent using natural resources as coagulant-flocculant agent has given positive impact on the growth of okra plants and is potentially utilized as biofertilizer for plants.


Introduction
Aquaculture is one of the fastest growing industries in food production worldwide and this industry includes a major source of protein for human nutrition.According to Department of Fisheries Malaysia [1], aquaculture production in Malaysia recorded a quantity of 412 metric tons equivalent to RM3.30 billion in 2019.Every production produces waste.Aquaculture industry wastes typically contain suspended solid (SS) consisting of unused inputs and by-products such as water supply, fish seeds and fertilizers in the form of effluent water [2].Aquaculture effluent can contribute to then environmental pollution if left untreated and unutilized.Therefore, effluent need to be treated before release to the environment.
2 Coagulation/flocculation process using inorganic coagulant/flocculant agents is among the most common and is traditionally used to treat wastewater with high SS [3].The most used salts are aluminium and iron (sulphate and chloride) [3].The use of chemicals in wastewater treatment processes raises a great deal of environmental and health issues such as Alzheimer's disease [4].In addition, the use of these chemical coagulant/flocculant agents will produce sludge that is toxic and not easily decomposed.
Hence, organic coagulant/flocculant agent have been introduced aggressively in recent years to substitute chemicals coagulant/flocculant agents in wastewater treatment processes due to the non-toxic sludge generated after treatment [5].Organic coagulant/flocculant agents are based on crustaceans [6], plants [7][8] and microorganisms [9].Ezemagu et al. [10] employed biocoagulant extracted from Tympanotonos fuscatus shell to treat petroleum produced water.The sludge produced after the biocoagulation treatment contains nutrients such as calcium, magnesium, phosphorus and sodium.Similar nutrients presence in sludge generated after treatment of water produced during the extraction of crude oil using biocoagulant extract from Terebralia palustris shell [11].The existence of nutrients in sludge produced after treatment indicates its potential in anaerobic digestion for biogas production [10][11].
In addition, sewage sludge containing nitrogen and phosphorus produced after treatment having the potential in becoming soil conditioner or fertilizer [12][13].The characterization of sludge after treatment process is important in order to determine its potential to be recovered and converted into valuable products.The characteristics of sludge produced after biocoagulation/flocculation of aquaculture effluent remain unknown.Therefore, the objectives of current study are to: (a) characterize the sludge produced after coagulation/flocculation process of aquaculture effluent using biocoagulant/flocculant agents and (b) investigate its effect on plant growth.

2.
Materials and methods

2.1
Origin of sludge Sludge used in this study is collected after coagulation-flocculation process of aquaculture wastewater.The aquaculture wastewater was obtained from a catfish farm in Negeri Sembilan, Malaysia.The concentrations of TSS, colour and nutrients in aquaculture wastewater collected was reported by Ahmad et al [2]: 427.7±3.1 mg/L (TSS), 1,310.7±2.5 ADMI (colour), 24.5±1.5 (ammonia nitrogen, NH3-N), 11.9±3.82(nitrate, NO3 -) and 0.07±0.1 mg/L (phosphate, PO4 3-).Biocoagulant agent extracted from plants was employed to treat aquaculture wastewater in order to reduce the TSS concentration.Sludge produced after this process was collected and dried under the sunlight.It was the grinded using an industrial grinder (Panasonic, Malaysia) and sieved with 255 nm sieve.

2.2
Analysis of nutrient content in sludge, garden soil and sand Bioavailable nutrient content in sludge, garden soil and sand were extracted before being analyzed.Calcium chloride (CaCl2) with 0.01 M was used in the extraction steps suggested by Houba et al. [14].Dried sample of sludge, garden soil and sand with a mass of 10 g were placed in a separate 250 mL conical flask.Then, 100 mL 1.0 M CaCl2 was added into the conical flask and agitated for 120 minutes using an orbital shaker at 130 rpm.Then, it was centrifuged for 10 min at approximately 3,000 rpm.Filtration through filter paper (porosity of 0.45 μm) (Whatman, Germany) was performed to obtain a clean supernatant sample.This sample was stored in a polyethylene container at 4 °C for further analysis using UV-spectrophotometer DR6000 (HACH Company, USA).Determination of nitrogen (N) in the form of NO3 -and phosphorus (P) PO4 3-content was carried out using HACH Method 8039 and Method 8048, respectively.The N and P contents in sludge were: 1.1 g N/g sludge and 0.5 g P/g sludge, garden soil: 5.2 g N/g garden soil and 0.4 g P/g garden soil and sand: 2.8 g N/g sand and 0.4 g P/g sand.

Preparation of growth medium
Three reed beds, each with 48 cm length × 19 cm width ×17 cm height, were used and filled with 5 kg of different growth medium; garden soil (GS), sand (S) and sand with sludge (SS).GS represents the nutrient rich medium and acts as positive control in this study.While sand was selected as nutrient deficiency medium and serve as negative control in this study.Concentration of N during characterization of sludge, garden soil and sand were used as the baseline to initiate the optimal mixture percentage ratio between sludge and sand.The formula used in the calculation is: with, [NGS] is the concentration of N in garden soil, [NS] is the concentration of N in sand, [NSludge] is the concentration of N in sludge, WGS is the weight of garden soil, WS is the weight of sand and WSludge is the weight of sludge.
After calculation using Eq. ( 1), the composition of planting medium for sand with sludge was 88% sand and 12% sludge.

2.4
Source and germination of Abelmoschus esculentus L. Seeds of A. esculentus L or okra (common name) used in this study is from Green World Genetics Sdn.Bhd.(Malaysia).Seed germination was done by placing the seeds in seed-starting soil mix.The soil was kept moist by watering them twice daily.After two weeks, saplings with 5-8 cm of height were grown.They were transferred to the reed beds with different growth medium to evaluate the growth performance on different nutrient availability.

2.5
Cultivation of A. esculentus L in different growth medium.Each of the plant growth site (GS, S and SS) was planted with three saplings of A. esculentus L. The planting period was about eight weeks.Watering process was done daily.The physical observation of plants was monitored weekly whereby the condition of plants such as healthy, withered, died, flowering and fruiting stage was observed and photographed.The definition for each observation was explained further in Table 1.Healthy plants are considered when all parts of the plant are green.When some parts of the plant turn yellow represent withered plants.Plants are considered died when all parts of the plant turn brown.On top of that, plants at flowering and fruiting stage shown when flowers start blooming and pods start producing for the first time, respectively.Additionally, the growth (number of leaves and height of plants) of A. esculentus L. was recorded weekly throughout the eight weeks of planting period.As for the dry weight of plants, it was only documented during week 0 and 8.The relative growth rate was calculated using the following formula [15] (2): (2) with, WW0 is the dry weight of plant (g) on week 0, WW8 is the dry weight of plant on week 8 and t is the planting period (56 days).
Experimental setup related to this study on the effect of sludge resulting from biocoagulationflocculation process on A. esculentus L. growth was represented in Table 2.

2.5
Statistical data analysis SPSS Statistics version 21 (SPSS Inc., USA) at 95% confidence levels [2] was utilized to analysed the data from this study.The experimental data including the numbers of leaves, plant height and concentration of nutrients were statistically judged using a two-way analysis of variance (ANOVA) while the dry weight and RGR were analysed using a one-way ANOVA.

Physical observation of A. esculentus L.
Table 2 shows the physical observations of plant conditions that occurred during the planting period.In overall, plants in all planting medium (GS, S and SS) were healthy throughout the 8 weeks of planting period.However, it is crystal clear that the growth of A. esculentus L. planted in sand during week 5 and 8 were shorter than plants planted in GS and SS.It was also observed that the plants in GS and SS already reached fruiting stage.The fruit is circled in red in Table 2. Plants in SS with two fruits while plant in GS appeared to have only one fruit.The application of various planting medium was found to cause the difference in plant growth.

Table 2
The physical plant growth of A. esculentus L. during week 1, 5 and 8 of planting period 3.2 A. esculentus L. growth Fig. 1(a) depicts the number of leaves for A. esculentus L. all through planting period of A. esculentus L. It can be seen from the figure, the number of plant leaves in GS and SS increased until the end of the study.But there is no significant difference between the number of plant leaves in GS and SS.In contrast, the number of plant leaves in SS is significantly higher at p<0.05 than the number of leaves for plants planted in the sand.
Fig. 1(b) provides an overview of the height of A. esculentus L. in all planting medium throughout the eight weeks of planting period.From the graph, it is apparent that the height of plants in all planting medium kept increasing until the end of study period.Plant height for plants in SS was significantly higher (p<0.05)than those in GS and S, during week 6 onwards and week 3 onwards, respectively.At the end of planting period, plants planted in sand was the shortest (30 cm) than plants in GS (86 cm) and SS (91 cm).Fig. 2. displays the results of the dry weight and relative growth rate (RGR) of plants.From the graph, it can be concluded that the dry weight (31.4 g) and RGR (3.8 day -1 ) of plants in SS at the end of planting period (week 8) were significantly higher at p<0.05 than the plants in S (dry weight= 1.9 g and RGR= 0.1 day -1 ), while it was not significantly higher at p>0.05 than the plants in GS (dry weight= 29.0 g and RGR= 3.5 day -1 ).
It can be concluded that the growth of A. esculentus L. planted in SS is approximately equal with the plants in GS.However, they experienced greater growth in SS than in sand.A possible explanation for this might be due to the presence of nutrients in the planting medium.The initial concentration of nutrients, nitrogen (N) and phosphorus (P) in the form of nitrate (NO3 -) and phosphate (HPO4 2-) respectively, will be discussed further in the next section.

3.3
Nutrient content in planting medium Looking at Fig. 3, it is apparent that the N and P in the form of NO3 -and HPO4 2-respectively, decreased with planting period.The initial concentration of NO3 -in SS (5.21 g N/g SS) is significantly higher (p<0.05)when compared in sand (2.83 g N/g S) while no significant difference (p>0.05) in GS.As for initial concentration of HPO4 2-, it is higher in SS compared in sand and GS but there is no significant difference (p>0.05) between them.Therefore, the presence of N and P could be a major factor causing the difference in the plant growth.This provides some explanation as to why the plants in SS were having greater growth than plants in sand while plants in SS and GS having nearly the same growth.

4.
Conclusions In this study, the effect of sludge produces after coagulation-flocculation process of aquaculture wastewater using plant-based coagulant on the growth of A. esculentus L. was observed.The sludge containing nutrient, nitrogen (N) and phosphorus (P), makes it an important candidate for its use as biofertilizer.The valuable effect has been tested positively on A. esculentus L., resulting in higher number of leaves, weight and plant height.Thus, it can be assumed that the N and P content presence in sludge can be a good source of plant nutrients and help to enrich nutrient deficiency planting medium.

Table 1
Physical observations of okra plants condition along the exposure period Observation Definition Picture represents physical observation Healthy All parts of the plant are green Withered Some parts of the plant turn yellow Died All parts of the plant turn brown Flowering stage Flowers start blooming Fruiting stage Pods start producing for the first time

Fig. 1 .
Fig. 1.Plant growth as for (a) the number of leaves and (b) the height of plant during eight week of exposure period.Asterisk symbol (*) represents significant difference at p<0.05 in data between planting medium (GS or S) when compared with SS

Fig. 2 .
Fig. 2. Dry weight and relative growth rate of plants.Asterisk symbol (*) represents significant different at p<0.05 in data between planting medium (GS or S) when compared with SS

Fig. 3 .
Fig. 3. Nutrient in planting medium for (a) nitrate and (b) phosphate during eight week of exposure period.Asterisk symbol (*) represent significant different at p<0.05 in data between planting medium (GS or S) when compared with SS.

Table 2
Planting medium involved in this study with respective to their role