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Fish Farm Wastewater Treatment using Moringa oleifera Seed Powder

as Natural Coagulant
To cite this article: Cheah Yi Tong et al 2021 IOP Conf. Ser.: Earth Environ. Sci. 945 012070

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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

Fish Farm Wastewater Treatment using Moringa oleifera

Seed Powder as Natural Coagulant
Cheah Yi Tong, Fakhira Huda Binti Che Yusuf and CJC Derek*
School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia,
14300 Nibong Tebal Penang, Malaysia

*Corresponding author’s e-mail: [email protected]

Abstract. In view of the huge amount of wastewater being generated from the aquaculture
sector in Malaysia, Moringa oleifera seed powder was utilized as effective bio-coagulant to
minimize the deterioration of fish farm wastewater quality. In this study, optimization of a
coagulation-flocculation process was examined through response surface model toolkit
established on a central composite design. A quadratic polynomial model was eventually
selected to fit its turbidity removal response. The effect of four crucial parameters, namely pH,
coagulant dosage, mixing time and settling time was optimized at pH 10, 400 mg/L coagulant
dosage, 15 minutes mixing time and 10 minutes settling time for M. oleifera seed extract at a
47.11% turbidity removal efficiency. M. oleifera seed powder substantially removed (>30%)
the concentration of total suspended solids, ammonia content, total volatile solids, and
phosphate content as compared with the control group without any coagulant addition.

Keywords: Moringa oleifera, Central Composite Design, Turbidity Removal, Fish Farm
Wastewater.

1. Introduction
Over the past few years, development of aquacultures as monocultures has manifested tremendous
growth, from keeping fish in ponds for easy harvesting to employing feed, hormones and antibiotics in
high-tech tech fish farms. Global aquaculture production has reached 106 million tons in 2015 alone,
with 76.7 million tons coming from aquatic animals while the rest is from aquatic plants, with average
growth percentage of 6.6 % since 1995 [1]. This positive trend is projected to continue as the
aquaculture sector plays a huge role in contributing to food security and poverty alleviation. However,
it is well known that aquaculture activities are the major cause to the elevation in concentration of both
organic waste and toxic compounds. These contaminants, particularly ammonium and nitrite, could be
harmful to aquatic life and induce “blue baby syndrome”. Moreover, extensive growth of either
macro- or micro-algae is stimulated, resulting in excessive eutrophication. Hence, it is of vital
importance to incorporate effective fish farm wastewater treatment in Malaysia.
In order to achieve high quality of receiving waters in aquaculture systems, broad range of
conventional methods including settling, centrifugation, filtration and biological processes have been
implemented. Coagulation/flocculation (CF) process was long-recognized as a fairly simple and cost-
effective alternative in wastewater clarification [2]. Chemical coagulation is typically used in this case,
with inorganic coagulants such as aluminum, iron, and magnesium salts being commonly employed.
However, the usage of chemical coagulants, in particular, could lead to production of sludge with high
toxicity because of its aluminum content, not to mention the presence of residual aluminum
concentration in the treated water which results in a big drop of wastewater pH. The exorbitantly
expensive cost of imported chemicals in some developing countries is also one of the drawbacks for its
application [3].
M. oleifera, a natural pantropical plant of monogeneric Moringaceae family, has been well-
recognized for its active bio-coagulate compounds. The property of M. oleifera that permits its ability
to function as a coagulant is due to the presence of cationic protein called ‘lectin’, which is an active
ingredient specifically found in its seed. Its coagulation ability has been well-demonstrated via the past
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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

researches in treating several types of wastewaters [4-6]. In general, CF treatment efficiency is

strongly dependent on the optimum operating factors to avoid colloids re-stabilization. Interruption of
the bridge formation between neighboring particles might occur, especially if the coagulant is
overdosed [7]. Nevertheless, factor-by-factor assessment is too time-consuming, especially
confronting with a broad range of manipulated variables. Therefore, this study aims to optimize a CF
condition to effectively remove the turbidity level in the fish farm wastewater using M. oleifera seed
powder.

2. Materials and Methods

2.1 Materials
The fish farm effluent was collected from Chop Aik Lee Fishery (GPS coordinate 5.1189,100.4164) in
Parit Buntar, Perak, which is around 10 km from the Engineering Campus, Universiti Sains Malaysia.
It was collected and held in a cold storage chamber before being analyzed. The M. oleifera seeds were
gathered and dried in the oven at 55 ˚C overnight. Using a grinder, the kernels were crushed and
grounded to medium fine powder, then followed by an addition of 40 mL distilled water into a beaker
containing 2 g powder. The mixture was continually agitated for 6 hours to extract the lectins, and
eventually ending up in a 2 % stock solution.

2.2 Coagulation-flocculation experiments

Coagulation activity of M. oleifera seed powder was examined using conventional jar test. Each
beaker contained 300 mL fish farm wastewater collected by rapid (250 rpm) and followed by slow (40
rpm) mixing. After observing two different separating layers, the supernatant was gathered and
subjected to pH, turbidity, ammoniacal nitrogen, chemical oxygen demand (COD), mixed liquor
suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS) and phosphate content
analysis. The main target response, turbidity was checked by a portable turbidity meter (HI 93703,
Hanna Instruments, Italy) while the ammoniacal nitrogen (N-NH3) concentration was examined by
Salicylate Method. COD was quantified by Dichromate/H2SO4 Method. For MLSS analysis, 30 mL
water samples was filtered through weighed standard glass microfiber filters (WhatmanTM, GF/C
filter, 0.45 μm, 47 mm in diameter) and remained residues on the filters were dried at 105˚C in oven
for 1 hour. For MLVSS analysis, the same filters were ignited at 550˚C in furnace for 20 min.
Phosphate content was measured using Ascorbic Acid Method (HACH method 8048).
All the obtained results were compared with the control (wastewater samples without addition of
any coagulant). The percentage removal was calculated according to the equation:
𝐶0 − 𝐶𝑖
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑒𝑚𝑜𝑣𝑎𝑙 (%) = × 100% (1)
𝐶0
where 𝐶0 is the initial concentration (mg/L) and 𝐶𝑖 is the final concentration (mg/L) for all the
parameters except turbidity which the units are in Nephelometric Turbidity Unit, NTU.

2.3 Design of experiments and statistical analysis

Empirical investigation on CF treatment on fish farm wastewater was performed by employing design
of experiment (DOE) method via Design Expert software version 7.1.5 (STAT-EASE Inc.,
Minneapolis, USA). The following important components involved were enumerated as follows: M.
oleifera dosage, X1 (400 to 1,200 mg/L), pH, X2 (4 to 10), mixing time, X3 (5 to 15 min), and settling
time, X4 (10 to 30 min).
Based on the four variables, the experimental models had 8 axial or star points (α= +1) at the center
and both extreme levels, as well as 6 replicates of the central point. As shown by the equations below,
total 30 experiments were calculated for M. oleifera seed powder.
𝑁 = 2𝑛 + 2𝑛 + 𝑛𝑐 (2)

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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

where N is the total number of experiments required, n is the number of factors and 𝑛𝑐 is the number
of replicates at the center point. The turbidity response was used to develop an empirical model using
a second-order polynomial equation as shown below:
𝑛 𝑛 𝑛−1 𝑛
𝑌 = 𝑏𝑜 + ∑ 𝑏𝑖 𝑥𝑖 + (∑ 𝑏𝑖𝑖 𝑥𝑖 ) + ∑ ∑ 𝑏𝑖𝑗 𝑥𝑖 𝑥𝑗 (3)
𝑖=1 𝑖=1 𝑖=1 𝑗=𝑖+1

where Y represents the predicted response, 𝑏0 is the constant coefficient, 𝑏𝑖 is the linear coefficients, 𝑏𝑖𝑗
is the quadratic coefficients and 𝑥𝑖, 𝑥𝑗 are the coded values of the process variables. Table 1 presents
the central composite design matrix applied of the experiments.

Table 1. Experimental design matrixes for M. oleifera seed powder.

Run Coagulant dosage, pH, Mixing time, Settling time, Turbidity,

X1 (mg/L) X2 X3 (min) X4 (min) Y1 (NTU)
1 1,200 7 10 20 268
2 1,200 10 5 30 266
3 1,200 4 5 30 285
4 800 7 15 20 266
5 400 10 5 10 258
6 800 7 5 20 262
7 400 4 5 30 243
8 800 10 10 20 258
9 800 7 10 20 256
10 400 4 15 30 234
11 1,200 10 15 30 216
12 400 10 15 10 238
13 400 7 10 20 228
14 1,200 10 15 10 246
15 800 7 10 20 213
16 400 4 5 10 235
17 800 7 10 20 231
18 400 10 5 30 234
19 800 7 10 20 230
20 1,200 10 5 10 245
21 800 7 10 20 221
22 800 7 10 20 164
23 400 4 15 10 102
24 1,200 4 5 10 124
25 400 10 15 30 79.2
26 1,200 4 15 10 153
27 800 7 10 10 120
28 800 7 10 30 134
29 1200 4 15 30 76.5
30 800 4 10 20 85.1

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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

3. Results and Discussion

3.1 Development of regression model equation and analysis of statistical significance

Based on the sequential model sum of squares, a second order polynomial mathematical equation with
linear and quadratic terms has been generated from the main response. The developed equation for M.
oleifera seed powder in terms of coded factors was shown by Equation 4.

Y1 = 230.92 - 12.07A - 73.57 B - 7.63C - 12.21AB - 7.71BC - 42.04B2 (4)

where A, B and C were the coded values of the process variables coagulant dosage, pH, and mixing
time for M. oleifera seed powder. It was noted that settling time was missing here as it was found
statistically insignificant. The value of predicted R2 for M. oleifera seed powder is 0.9357 while the
adjusted R2 is 0.9730. It was demonstrated that the quadratic model could be adjusted to the
experimental data satisfactorily. As depicted in Table 2, F-value of 2.74 implied the lack of fit is not
significant, suggesting a great model fitting.

Table 2. ANOVA analysis for jar test using M. oleifera seed powder.

Source Sum of Squares df Mean Square F- Value Prob > F

Model 1.17E+05 6 19524.33 135.24 < 0.0001
A – Coagulant Dosage 2623.29 1 2623.29 18.17 0.0003
B – pH 97416.98 1 97416.98 674.77 < 0.0001
C - Mixing Time 1047.29 1 1047.29 7.25 0.013
AB 2383.88 1 2383.88 16.51 0.0005
BC 950.18 1 950.18 6.58 0.0173
B2 12724.33 1 12724.33 88.14 < 0.0001
Residual 3320.52 23 144.37
Lack of Fit 3015.19 18 167.51 2.74 0.1339
Pure Error 305.33 5 61.07
Cor Total 1.21E+05 29

Model validity was further investigated by observing the clustering pattern of all the data points. As
shown in Figure 1, points were scattering randomly along a 45°regression line, indicating small
deviation between the actual and predicted values. In other words, this current model was accurate
enough, thus no additional model revision was required.

Figure 1. Predicted vs. actual for M. oleifera seed powder.

3.2 RSM predictions and the influence of process variables towards the turbidity removal
efficiency
From the 3D response surface plots plotted based on the predicted model Equation 4, pH was the
most important parameter, then followed by coagulant dosage and mixing time. As indicated by
ANOVA, the settling time was insignificant (p>0.05). Figure 2(a) signified that turbidity tended to be
decreased when the pH was rise until pH 10. The optimum pairing of the operating parameters were
eventually predicted at coagulant dosage of 400 mg/L, 15 min of mixing time, and 15 min of settling

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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

time. In Figure 2(b) and (c), a noteworthy observation here was the continuous decrease in turbidity
value though coagulant dosage was maximized at 1,200 mg/L. It was postulated that the critical
coagulation point was not yet achieved at this specific dosage amount, hence higher amount of
coagulant could be given a try. As compared with dominant pH factor, both mixing time and settling
time were insignificant as displayed in Figure 2(d) and (e). However, mixing time was playing a more
dominant role over settling time with the turbidity reduction as shown in Figure 2(f).

(a) (b) (c)

(d) (e) (f)

Figure 2. Response surface plot showing the interaction between two parameters: (a) pH and
coagulant dosage, (b) coagulant dosage and mixing time, (c) coagulant dosage and settling time, (d)
pH and mixing time, (e) pH and settling time, and (f) mixing time and settling time using M. oleifera
seed powder as the natural coagulant.
Each coagulant has its own critical inflection point, beyond which the aggregated particles would
be re-dispersed again, causing the disruption in particle settling [8]. If the coagulant dosage is
exceeded, water turbidity could be deteriorated because extra coagulants no longer interact with
oppositely charged colloidal particles after most of the colloids have been neutralized and precipitated
[5]. Similarly, an optimum acidity level has been well known for its remarkable influence towards the
CF process as the alkaline amino acids from M. oleifera protein were able to ionize and produce
carboxylate ion and proton. Subsequently, colloids carrying electrons were attracted to form a neutral
group with strong attractive forces among each other, leading to stabilized aggregates [5]. Due to the
isoelectric point of the proteins in the seeds ranging from 10 to 11, coagulation performance of M.
oleifera seed powder is known to be efficient at alkaline condition [9]. On the other hand, increasing
mixing time has a minor influence on turbidity reduction, indicating that the collision rate was no
longer preponderant if there was insufficient coagulant dosage. Flocs formed were not dense enough

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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

to settle quickly though given a prolong settling time. In such context, the flocs would be most
probably broken up again due to the rapid mixing time which brings extensive shear force towards the
loosely organized flocs.

3.3 Optimization of coagulation/flocculation process

The optimization of CF process was performed by minimizing both the coagulant dosage and settling
time from a cost-effective perspective, while both mixing time and pH value was defined to be within
the range with the aim of highest coagulation performance. According to the highest desirability
proposed from the software, optimization solution has demonstrated that fish farm wastewater was
having a high potential to be treated by M. oleifera seed powder at 400 mg/L coagulant dosage, pH 10,
15 min mixing time and 10 min settling time with a predicted turbidity value of 128.854. A similar
optimized CF process was then carried out to verify the model prediction. As a result, real
experimental value obtained was totally in agreement with the predicted value with only 0.66 % small
difference.
Moreover, pre- and post- treated fish farm wastewater quality was also characterized as shown in
Figure 3. Majorly, all the parameters were experiencing a significant increase in their respective
percent removal for M. oleifera seed powder, with the only exemption on ammoniacal nitrogen and
COD. Generally, leftover feed and biological metabolites of organisms were the predominant sources
of N-containing compounds in aquaculture waters. M. oleifera seed powder was not as effective as the
control in removing ammonia, with a removal efficiency of just around 30 % and significantly less
than the control (56 %). The ammonia concentration in the finished product was still well above the
acceptable discharge limit. The water samples would need to undergo supplementary physico-
chemical treatment or increase the coagulant dosage to guarantee that the final concentration of TSS
meets the discharge limit stipulated by the Environmental Quality Acts of 1974. The results obtained
from M. oleifera in this study differ with those obtained from other researchers who reported an
increase in phosphate content [9]. This is due to the fact that M. oleifera's crude water extract contains
a considerable amount of orthophosphates, which could be present as leachates in the wastewater
samples, as well as differing wastewater quality from the previous researches.

70
Control M. oleifera seed powder
Removal percentage (%)

60
50
40
30
20
10
0
Turbidity Ammoniacal COD TSS TVS Phosphate
nitrogen content
Fish farm wastewater quality component
Figure 3. Comparison of water quality characteristics between control sample and samples using M.
oleifera seed powder.

4. Conclusion
M. oleifera seed powder was found to be an excellent bio-coagulant in the treatment of fish farm
effluent from aquaculture farm in Malaysia. The design of a reliable response surface model has
proven to well-estimate the relative turbidity removal response. For the jar test utilizing M. oleifera
seed powder, the quadratic model created in this work had a R2 value of 0.9860, indicating a quadratic
regression model with high significance level. The turbidity result for M. oleifera seed powder was

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4th International Symposium on Green and Sustainable Technology (ISGST 2021) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 945 (2021) 012070 doi:10.1088/1755-1315/945/1/012070

well matched with only 0.66 percent error after optimization of relevant process parameters. This
study concluded that M. oleifera seed powder has the potential to provide moderately efficient
wastewater treatment in fish farms. Future pilot scale experiments using M. oleifera seed powder as a
bio-coagulant could be used for fish farm effluent remediation in order to meet the environmental
legislation's discharge limits.

Acknowledgements
This work is supported by the Long Term Research Grant Scheme (LRGS) LRGS/1/2018/USM/01/1/2
(UTAR/4411/S01) (PJKIMIA/6770007) by Ministry of Higher Education, Malaysia.

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