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 http://www.iaeme.com/IJCIET/index.asp 967 editor@iaeme.com 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]. http://www.iaeme.com/IJCIET/index.asp 968 editor@iaeme.com 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 Land spreading Incineration Treatment (Stabilization) Elaborations References Advantages: High nutrient contents have advantageous impacts on the physical condition of land and productivity of crop. Disadvantages: Odor problem and increase the risk of contamination of groundwater. Advantage: Dispose large amount of sludge in one time. Disadvantage: Smoke and ash produced will cause pollution and it is expensive to operate and maintain. Carried out through biological, chemical or thermal process. Biological process is highly suggested as it requires low costs and creates fewer issues on environment and operation. [14, 15] [16,17] [17,18] 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. http://www.iaeme.com/IJCIET/index.asp 969 editor@iaeme.com Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim Ghani Table 2 Applications and Uses of Eco Enzyme [21, 27] Fields Household Applications Uses Household cleaner, detergent, body care, car care Air purification and deodorizer Food preservation Agriculture, Aquaculture and Animal Husbandry Insecticide Organic fertilizer and pesticides Treatment Catalyst Removes impurities on the surface. Removes odour and dissolved toxic air. Prevent the growth of microorganisms to preserve the foods. Keep the farm free from insects and infections. Increase crop yields without pollutions. Decompose, compose and transform the organic matters. 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 TS TDS BOD COD Citric Acid Protease Activity Amylase Activity Lipase Activity pH meter Apha Standard Method 2540 B Apha Standard Method 2540 C Apha Standard Method 5210 B Apha Standard Method 5220 C High Performance Liquid Chromatography (HPLC) Method Casein Digestion Unit (CDU) Analytical Method 3,5-dinitrosalicyclic Acid (DNS) Method 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. http://www.iaeme.com/IJCIET/index.asp 970 editor@iaeme.com 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 TDS (mg/l) TS (mg/l) BOD5 (mg/l) COD (mg/l) Citric Acid (mg/l) 2.79 14,000 17,000 40 80,000 14,130 2.86 14,000 19,000 33 96,000 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 http://www.iaeme.com/IJCIET/index.asp 971 editor@iaeme.com 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]. http://www.iaeme.com/IJCIET/index.asp 972 editor@iaeme.com 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) VSS (mg/l) TAN (mg/l) TP (mg/l) 3,067 989 118 218 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]. http://www.iaeme.com/IJCIET/index.asp 973 editor@iaeme.com Nazaitulshila Rasit, Lim Hwe Fern and Wan Azlina Wan Ab Karim Ghani 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% http://www.iaeme.com/IJCIET/index.asp 974 editor@iaeme.com 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 http://www.iaeme.com/IJCIET/index.asp 975 editor@iaeme.com 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 http://www.iaeme.com/IJCIET/index.asp 976 editor@iaeme.com 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. 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