International Journal of Civil Engineering and Technology (IJCIET)

Volume 10, Issue 03, March 2019, pp. 967-980. Article ID: IJCIET_10_03_094
Available online at http://www.iaeme.com/ijciet/issues.asp?JType=IJCIET&VType=10&IType=03
ISSN Print: 0976-6308 and ISSN Online: 0976-6316

© IAEME Publication Scopus Indexed

PRODUCTION AND CHARACTERIZATION OF

ECO ENZYME PRODUCED FROM TOMATO
AND ORANGE WASTES AND ITS INFLUENCE
ON THE AQUACULTURE SLUDGE
Nazaitulshila Rasit
School of Ocean Engineering, Universiti Malaysia Terengganu (UMT), 21030 Kuala Nerus,
Terengganu, Malaysia

Lim Hwe Fern

School of Ocean Engineering, Universiti Malaysia Terengganu (UMT), 21030 Kuala Nerus,
Terengganu, Malaysia

Wan Azlina Wan Ab Karim Ghani

Department of Chemical Engineering, Faculty of Engineering, Universiti Putra Malaysia
(UPM), 43400, Serdang, Selangor Darul Ehsan, Malaysia.

ABSTRACT
Eco enzyme is a fermented solution from mixture of sugar, fruit wastes and water
in the ratio of 1:3:10. It can be used as pretreatment for aquaculture sludge before
other further treatments in order to minimize various environmental problems that
caused by high nutrient contents of the sludge. In this study, the characteristics of the
eco enzymes (tomato and orange wastes) were investigated and its potential efficacy of
parameters removal within 10 days treatment was also evaluated. Results showed the
enzymes were acidic, composed of high total solids (TS), total dissolved solids (TDS),
biological oxygen demand (BOD), chemical oxygen demand (COD), citric acids and
contained biocatalytic enzyme activities which are protease, amylase and lipase. The
enzymes were also showed a higher removal percentage of total suspended solids (TSS),
volatile suspended solids (VSS), total phosphorus (TP), total ammonia nitrogen (TAN)
and COD approximately 87%, 67%, 99%, 91% and 77% respectively in the optimum
concentration of eco enzymes (10%). Besides, use of orange eco enzyme (OEE) was
showed higher removal percentage compared to tomato eco enzyme (TEE) due to high
organic acid contents. These findings may contribute to the effectiveness of enzymatic
pretreatment which can reduce the inhibitors and improve further treatment processes
in treating other industrial wastes.
Keywords: Eco enzyme, Aquaculture sludge, Orange eco enzyme, Tomato eco
enzyme

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 Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim Ghani

Cite this Article: Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim
Ghani, Production and Characterization of Eco Enzyme Produced from Tomato and
Orange Wastes and Its Influence on The Aquaculture Sludge, International Journal of
Civil Engineering and Technology, 10(3), 2019, pp.967-980.
http://www.iaeme.com/IJCIET/issues.asp?JType=IJCIET&VType=10&IType=03

1. INTRODUCTION
Aquaculture is one of the industries that being growing fast in Malaysia due to the increment
of human population and directly increase the number of fish demand for human consumption
[1]. The output is increased up to 141,837 tonnes in 13 years when started in year 1990 [2].
Therefore, it is expected that the fish production will be increased of more than 60% in the
future by practicing variety of aquaculture cultural methods to culture the aquatic organisms in
three types of environment which are freshwater, marine and brackish water. Then, the rapid
growth of aquaculture had also increased the Malaysia incomes by exporting those productions
to other countries [3, 4].
Basically, large amount of effluents and sludge were produced from aquaculture activities
in the industry. It is known that the major cause of sludge formation is from animal feeding
due to the feeding activities that will induce the sludge formation [5, 6]. Aquaculture sludge is
also known as non-toxic agriculture waste due to its contents. The contents such as animals’
excrement and the decaying materials cause the sludge consists of total suspended solid (TSS),
total dissolved solid (TDS) and biological oxygen demand (BOD). Besides, the sludge also
contains total solid (TS), chemical oxygen demand (COD) and nutrients of nitrogen and
phosphorus causing by the unconsumed foods [7, 8]. Therefore, these contents will lead to
environmental problems such as water pollution even though it is characterized as a non-toxic
waste.
In order to minimize the impacts, various methods have been applied in the treatment of
aquaculture sludge as shown in Table 1. The first method is land application of sludge as
fertilizers to improve the soil condition and crop quality. The sludge can either directly applies
on the land or after some treatments, but no matter, it will cause the odor problem, increase the
costs of sludge management and lastly cause serious groundwater contamination since it still
contains those organic matters [9, 10, 11]. Besides, the incineration method had also limited by
these problems [12]. Hence, treatment by biological process on aquaculture sludge has been
widely since it requires low costs and fewer environmental issues [13].
However, biological processes such as aerobic and anaerobic digestion (AD) will be
inhibited by the high concentration of solids and nutrients that presented in the sludge [13, 19].
Therefore, commercial enzymes have been used as pretreatment because it possesses various
activities that can digest specific molecules of pollutants [20]. The addition of enzymes in
wastewater treatment can produce less toxic insoluble products, so that it can be eliminated
conveniently from the water bodies. But these commercial enzymes are too expensive and not
economical if apply on any treatment [21]. Thus, eco enzyme has been introduced to replace
the commercial enzyme. Eco enzyme is also known as garbage enzyme which was invented by
Dr. Rosukon from Thailand. It called as eco enzyme because it is produced from pre-consumer
supermarket residues such as vegetables, fruits or its peels which are abundantly available in
Malaysia. It is a dark brown liquid product that full of fruit fragrance or pungent vinegary smell
[22, 23, 24, 25].

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 Production and Characterization of Eco Enzyme Produced from Tomato and Orange Wastes and
Its Influence on The Aquaculture Sludge

Table 1 Methods for Aquaculture Sludge Treatment and Disposal from Previous Studies
Methods Elaborations References
Advantages:
High nutrient contents have advantageous impacts on
the physical condition of land and productivity of crop.
Land spreading [14, 15]
Disadvantages:
Odor problem and increase the risk of contamination
of groundwater.
Advantage:
Dispose large amount of sludge in one time.
Incineration Disadvantage: [16,17]
Smoke and ash produced will cause pollution and it is
expensive to operate and maintain.
Carried out through biological, chemical or thermal
process.
Treatment [17,18]
(Stabilization) Biological process is highly suggested as it requires
low costs and creates fewer issues on environment and
operation.
Eco enzyme has numerous uses and applications in different fields as shown in Table 2. Its
functions are divided into four major groups which are decompose, compose, transform and
catalysis. Firstly, the eco enzyme can be utilized in household purposes such as cleaner to
remove the impurities on the surface because of its acidic condition. Next, it can equalize the
pollutants especially toxins in the atmospheres, water bodies or lands. Therefore, it can be used
as air purification or deodorizer as stated in the table in order to remove odor and dissolved
toxic air. Another application of eco enzyme is used as food preservation due to its propionic
acid content that effective in preventing the growth of microbes. The acetic acid within eco
enzyme can also destroy the organisms, so sometimes it can be used as insecticide or pesticide
[23, 26, 27]. Furthermore, the nitrate (NO3) and carbonate (CO3) contents in eco enzyme make
it used as a natural organic fertilizer. This is because the contents can improve the soil fertility
and directly increase the crop yields without any pollution. In treatment field, it is acts as a
catalyst because it has been used to speed up the decomposition, composition and
transformation of organic matters into simpler and safer substances [21, 23, 26].
This study will focus on production of eco enzymes produced from tomato and orange
residues and then characterization based on pH, TS, TDS, BOD, COD, citric acid concentration
and enzyme activities which are protease, amylase and lipase. It will also identify the effect
and efficiency of the fermented eco enzyme on contaminants removal from aquaculture sludge
by evaluating parameters such as TSS, volatile suspended solids (VSS), total phosphorus (TP),
total ammonia nitrogen (TAN) and COD before and after the 10 days treatment.

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 Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim Ghani

Table 2 Applications and Uses of Eco Enzyme [21, 27]

Fields Applications Uses
Removes impurities on the
Household cleaner, detergent, body surface.
Household care, car care
Air purification and deodorizer Removes odour and dissolved
toxic air.
Food preservation Prevent the growth of
microorganisms to preserve the
foods.
Agriculture, Keep the farm free from insects
Aquaculture Insecticide and infections.
and Animal Organic fertilizer and pesticides Increase crop yields without
Husbandry pollutions.
Decompose, compose and
transform the organic matters.
Treatment Catalyst
Speed up the reactions within
the treatment.

2. MATERIALS AND METHODS

Two bottles of eco enzyme solution with different fruit types were prepared in this study. The
ingredients needed for the fermentation of eco enzyme are including fruit, sugar and water.
Firstly, the orange that collected from pre-consumer supermarket residues were mixed with the
sugar and water in the 3:1:10 ratio where stands for 900 g of orange residues, 300 g of sugar
and 3000 g of water. The mixture was then poured in an air-tight plastic container that able to
expand and fermented for 3 months. The gases produced were then released after a month of
fermentation, so the cap of containers was slightly opened to release the pressure in order to
avoid rupturing. Lastly, these containers were situated in a cool, dry and well-ventilated area
for 3 months to produce the eco enzyme solution [17, 21].

Table 3 Parameters and Methods for Eco Enzyme Characterization [28, 29, 30, 31]
Parameters Methods
pH pH meter
TS Apha Standard Method 2540 B
TDS Apha Standard Method 2540 C
BOD Apha Standard Method 5210 B
COD Apha Standard Method 5220 C
Citric Acid High Performance Liquid Chromatography (HPLC) Method
Protease Activity Casein Digestion Unit (CDU) Analytical Method
Amylase Activity 3,5-dinitrosalicyclic Acid (DNS) Method
Lipase Activity Titrimetric Method
After 3 months of fermentation, the enzyme solutions were filtered and separated from fruit
residues. The characteristics of tomato and orange enzyme solution were analysed by using
different standard methods as shown in Table 3. The parameters are including pH, total solids
(TS), total dissolved solids (TDS), biochemical oxygen demand (BOD), chemical oxygen
demand (COD), citric acid concentration and enzyme activities which are protease, amylase
and lipase.

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 Production and Characterization of Eco Enzyme Produced from Tomato and Orange Wastes and
Its Influence on The Aquaculture Sludge

Aquaculture sludge was collected at QZA Freshwater Fish Farm which located at Kuala
Terengganu, Terengganu. After that, batch test was conducted to determine the effective
dosage of both eco enzyme solutions for the treatment of aquaculture sludge. Hence, beakers
were filled with sludge sample and different concentration of enzyme solution dilution (5, 10,
15 times dilution). The mixtures were then shielded with aluminium foil and allowed to digest
for 10 days. After that, 5 parameters namely total suspended solids (TSS), volatile suspended
solids (VSS), total phosphorus (TP), total ammonia nitrogen (TAN) and COD were analysed
before and after the treatment with its standard methods [29]. All the tests were replicated for
3 times to ensure the validity of the results.

3. RESULTS AND DISCUSSIONS

3.1. Characterization of Tomato and Orange Eco Enzyme
Table 4 displays the characteristics of tomato eco enzyme (TEE) and orange eco enzyme (OEE)
after 3 months fermentation. From the table, it is observed that both eco enzymes were acidic
with the low pH values which were 2.79 for TEE and 2.86 for OEE. Organic acid is an
important key in determination of acidity. This means that the higher the organic acid contents,
the lower the pH value. Thus, the eco enzymes had low pH values in this study as a result of
the high various organic acid contents such as acetic or citric acids [26, 33]. The other
parameters of OEE were then slightly higher than TEE except for 5-day biological oxygen
demand (BOD5) value. The BOD5 value of TEE was 40 mg/l whereas OEE was 33 mg/l.
Moreover, OEE was high in COD with the value 96,000 mg/l while COD value of TEE was
80,000 mg/l. High BOD5 and COD values indicated the enzyme solution contained large
amount of organic matters which due to the fruit wastes and brown sugar that being added as
substrate in fermentation process [17, 34]. These fermentation materials that considered as
organic solid wastes were also the factor for high TS and TDS in eco enzyme [35]. Both eco
enzymes were also same in TDS value of 14,000 mg/l and OEE contained 19,000 mg/l of TS
and 17,000 mg/l for TEE.

Table 4 Characteristics of Tomato and Orange Eco Enzyme

Parameters Tomato Eco Enzyme Orange Eco Enzyme
pH 2.79 2.86
TDS (mg/l) 14,000 14,000
TS (mg/l) 17,000 19,000
BOD5 (mg/l) 40 33
COD (mg/l) 80,000 96,000
Citric Acid (mg/l) 14,130 35,281
However, the findings in this study were different from the results in the previous studies
because of the fermentation time. As stated in a research by Nazim and Meera (2013), the
parameter values except pH will reduce over the fermentation time due to the degradation of
organic matters (OMs) by microorganisms presented in the enzyme solutions [36]. Therefore,
these results obtained were higher since the parameters were analysed immediately after 3
months fermentation. Besides, materials used for fermentation will also affect the results. The
study by Arun and Sivashanmugam (2015) had showed that the parameter values will be lower
if using molasses sugar compared to brown sugar [17]. This is because molasses sugar is the
unwanted substances from sugar production that contain microorganisms. Due to the presence
of these microorganisms, the organic matter in fruit wastes will be further decayed and cause
low value results. In this study, brown sugar was used in eco enzyme making, so high readings

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 Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim Ghani

were obtained [17, 37]. Furthermore, the fruit wastes other that its peel also contains the citric
acids which had contributed to the high acid values as shown in Table 3.1 [38].
Instead of having high organic contents, the uniqueness of eco enzymes to be applied as
treatment agent were the content of biocatalytic enzyme activities which are lipase, amylase
and protease. The optimum pH for each enzyme activity is different. In general, the activities
may slow down, or enzymes become denature when out of the ranges of optimum pH due to
alteration of enzymes’ active sites by breaking its intermolecular bonds [17, 39].
Figure 1 presents the lipase activities of TEE and OEE at different pH values. It revealed
that OEE had higher lipase activity than TEE due to citric acid contents. This is because high
citric acids as contained in OEE will stimulate the cell lysis to release more intracellular
enzymes that carry out the activities [40]. Thus, higher enzyme activities were found in OEE.
Figure 1 was also showed that lipase activities for both enzymes were increasing started from
original pH value (pH 2.8) and reached its maximum activity (330 µ/ml for TEE and 690 µ/ml
for OEE) at pH 8. These results were similar to a research by Selvakumar and Sivashanmugam
(2017) that claimed the maximum lipase activity normally occurs at pH 8 and will keep
increasing until pH 9 [35].

Figure 1 Lipase Activity at Different pH Values

Smitha (2010) mentioned that the higher catalytic property of amylase will be occurred by
maintaining the pH values ranged from 6 to 7 [41]. Referring to Figure 2, both enzymes
contained high amylase activities within the pH range stated where the maximum activity was
occurred at pH 6.5 for TEE with the value of 2.62 µ/ml. While, OEE possessed 2.37 µ/ml as
maximum activity at pH 7.0.

Figure 2 Amylase Activity at Different pH Values

Figure 3 is demonstrating the higher protease activities for both enzymes were achieved
within the pH ranges of 6.5 to 7.5. However, the activities were greatly decreased at low pH
and pH higher than 7.5 because its optimum pH is between the ranges 6.5 to 7 [17].

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 Production and Characterization of Eco Enzyme Produced from Tomato and Orange Wastes and
Its Influence on The Aquaculture Sludge

Figure 3 Protease Activity at Different pH Values

3.2. Characteristics of Aquaculture Sludge
The characteristics of aquaculture sludge are tabulated in Table 5. The table shows the
aquaculture sludge comprised of high TSS and VSS with the values 3,067 mg/l and 989 mg/l,
respectively. On top of that, it’s also contained 118 mg/l of TAN and 218 mg/l of TP. These
high values were due to the animals’ excrement and unconsumed foods such as bread and
commercial fish feeds that being fed to the tilapia, freshwater lobsters and catfish [42, 43].

Table 5 Characteristics of Aquaculture Sludge

Parameters Aquaculture Sludge
TSS (mg/l) 3,067
VSS (mg/l) 989
TAN (mg/l) 118
TP (mg/l) 218

3.3. Pretreatment of Aquaculture Sludge in Batch Test

Pretreatment of aquaculture sludge for 10 days by using the different time dilutions of TEE and
OEE (5%, 10% and 15%) was conducted. During the 10 days treatment, five parameters such
as COD, TAN, TP, TSS and VSS were analysed for every 2 days. The degradations of those
parameters are presented in Figure 4 to Figure 8.
By observing the results in Figure 4, the removal percentage of COD in the sludge for both
eco enzymes was rose gradually once the eco enzymes were added. The maximum removal
percentage reached 76% for 10% of OEE, followed by 15% of OEE and 10% of TEE with the
same removal of 71%. This increasing trend was same as TSS and VSS removals which are
illustrated in Figure 5 and 6. The figures showed the highest TSS removal percentage was 87%
and 66% for VSS removal when treated with 10% of OEE. In general, low value of COD
indicates fewer organic matters in the sample [44]. Therefore, the COD can be reduced as the
biocatalytic enzymes were able to reduce TSS and VSS in the sludge. The reason for these
trends was due to citric acid contents of fruit wastes. The citric acids not only interfere the
extracellular polymeric substances’ (EPS) activities, but also release more enzymes by cell
lysis to stabilize the OMs. Generally, the EPS are found in aquaculture sludge and it will bind
with cations presented in the wastes to form flocs. Hence, the citric acid will disturb the binding
and then discarding the cations from the wastes to prevent the flocs formation [45, 46, 47].
This finding can be further explained by Kavitha et al. (2014) in which the enzymes are then
used to stabilize OMs into more soluble substances which will be decayed through anaerobic
digestion or further treatment easily in order to produce biogas such as methane and carbon
dioxide [20, 40, 48].

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As depicted in the Figure 4, 5 and 6, it can also be concluded that OEE treatment resulted
in higher removal percentages than TEE treatment. As mentioned in the earlier section, the
content of citric acids in OEE (35,281 mg/l) was far higher than TEE (14,130 mg/l), so more
enzymes were produced by citric acids to decay the OMs and reach higher removal percentage
[40].

Figure 4 Removal Percentage of COD in Aquaculture Sludge with Time for TEE and OEE dilutions
of 5%, 10% and 15%

Figure 5 Removal Percentage of TSS in Aquaculture Sludge with Time for TEE and OEE dilutions of
5%, 10% and 15%

Figure 6 Removal Percentage of VSS in Aquaculture Sludge with Time for TEE and OEE dilutions
of 5%, 10% and 15%

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 Production and Characterization of Eco Enzyme Produced from Tomato and Orange Wastes and
Its Influence on The Aquaculture Sludge

Similar to COD, TSS and VSS removals, TAN and TP were being removed started from
the first day of treatment period for each enzyme solution. As for TAN removal shown in the
Figure 7, the highest percentage was reached which is 90% when treated with 10% dilution of
OEE. Moreover, treatment by using 10% dilution of OEE also showed the highest TP removal
in the Figure 8 with the percentage of 99 whereas the lowest value only 95% in which treated
with 5% of TEE. For TP were removed by accumulating phosphorus (P) in the cells of
polyphosphate accumulating organisms (PAOs) in form of polyphosphate. During the process,
acetate ions from the eco enzymes as the carbon sources will form polyhydroxyalkanoates
(PHAs) to provide energy for PAOs to store P and then eliminate it from the sample [21, 49,
50]. Thus, higher acetic acids in OEE resulted in higher removal percentage of TP [51]. While,
the reason for the increment of TAN removal was due to the presence of organic acids. A
similar result was reported by Nazim and Meera (2013) in which the organic acids were helped
in breakdown of OMs into biogas and water [36].

Figure 7 Removal Percentage of TAN in Aquaculture Sludge with Time for TEE and OEE dilutions
of 5%, 10% and 15%

Figure 8 Removal Percentage of TP in Aquaculture Sludge with Time for TEE and OEE dilutions of
5%, 10% and 15%

3.4. Efficiency of Aquaculture Sludge Pretreatment using Tomato and Orange

Eco Enzymes
The removal percentages of various parameters in aquaculture sludge after 10 days treatment
with different time dilutions of TEE and OEE are shown in Figure 9 and 10. From the figures,
10% dilution of TEE and OEE were showed higher removal percentage for all the parameters

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 Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim Ghani

compared to 5% and 15% dilutions. The former was reduced approximately 71% of COD, 72%
of TAN, 98% of TP, 83% of TSS and 60% of VSS whereas the latter was removed these
parameters roughly 76%, 90%, 99%, 87% and 66% respectively. Hence, 10%-time dilution
was known as optimum concentration for achieving maximum removal potential. Based on
previous studies, the treatment of synthetic greywater by using 10% concentration of eco
enzyme was found to be more effective in removing TAN and TP [21, 36]. The research by
Tang and Tong (2011) was also showed higher TAN and TP removals with 9% dilution time
of eco enzyme in the wastewater [27].

Figure 9 Removal Percentage of Various Parameters in Aquaculture Sludge with time for 5%, 10%
and 15% dilutions of TEE

Figure 10 Removal Percentage of Various Parameters in Aquaculture Sludge with time for 5%, 10%
and 15% dilutions of OEE

4. CONCLUSION
In a nutshell, the growing human population had greatly influenced the aquaculture industry in
Malaysia. While developing the industry, not only the human fish consumption is keep
increasing, but also the wastes generated that may pollute the environment. Hence, there is a
need to reduce the impacts of wastes produced through pretreatment by eco enzyme. In this
study, eco enzymes produced from tomato and orange have been used as the pretreatment
method because it is more effective and low cost compared to commercial enzymes. Both eco
enzymes produced were found to be acidic and contained high amount of TS, TDS, BOD,
COD, citric acids and also biocatalytic properties of protease, amylase and lipase which react
as degradation factors of selected pollutants. Due to the characteristics and biocatalytic
property of eco enzyme, the eco enzymes were showed higher removal percentages of the

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 Production and Characterization of Eco Enzyme Produced from Tomato and Orange Wastes and
Its Influence on The Aquaculture Sludge

parameters especially TP and TAN which achieved up to 90% removal. Other parameters such
as total TSS, VSS and COD were removed roughly 87%, 67% and 77% respectively in the
sludge with the optimum concentration (10% times dilution) of eco enzymes. The higher
removal percentages of parameters were the results of high citric acids in eco enzyme. This
was also the reason for higher substance removals by using OEE compared to TEE. Therefore,
this study had proved the eco enzymes that produced from fruit wastes have potential to remove
the substances presented in the sludge. Then, these findings can contribute to the effectiveness
of pretreatment and then apply on other industrial wastes after investigating its characteristics.
The results can also enhance the efficiency of anaerobic digestion or other further treatments
and provide a solution for reducing the environmental problems. Further studies are still
required to figure out the suitable biological additives for those parameters’ removals
especially TSS, VSS and COD in the wastewater or sludge.

5. ACKNOWLEDGEMENTS
The authors wish to thank and acknowledge the Fundamental Research Grant Scheme (FRGS-
Vot. No. 59437), Ministry of Higher Education Malaysia for the financial support and
Universiti Malaysia Terengganu for its assistance provided during the course of this study.

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