Effects of Supplementing Effective Microorganisms (EM) to a Diet on Growth and Survival of Juvenile

Asian Seabass Lates calcarifer

R B Kennedy Enis*

Abstract

EM is an abbreviation for effective microorganisms and has been used around the world because EM’s applications are easy and harmless. The study on effects of supplementing EM to a diet on growth and survival of juvenile Asian seabass, Lates calcarifer was conducted under freshwater re-circulating systems in aquaria tanks for 28 days. Water quality parameters were standardized to be considered favorable for these fishes and temperature was set between 28-32oC. Juveniles were fed with control diet (G1) and treatment diet (G2) twice a day with feeding ration of 5%.d-1 and weighed every week. Specific growth rate (SGR) was 3.24 and 3.52%.d-1 between fish fed with control diet and fish fed with treatment diet respectively, and there was no significant difference in the growth between the two groups of fish. The survival rate for both experiments was 100%.

Keywords: Asian seabass, EM, Effective microorganisms, Lates calcarifer

1. Introduction

Effective Microorganisms, known as EM Technology, is a trademarked term. It is commonly used to describe a proprietary blend of three or more types of predominantly anaerobic organisms that was originally marketed as EM•1 Microbial Inoculant. But now, it is marketed by a plethora of companies under various names, each with their own proprietary blend. "EM Technology" uses a laboratory cultured mixture of microorganisms consisting mainly of lactic acid bacteria, purple bacteria, and yeast which co-exist for the benefit of whichever environment they are introduced, as has been claimed by the various EM-like culture purveyors. It is reported to include:

* Corresponding author. Tel.: +60 14 872 8882. E-mail address: rb_kent@yahoo.com © 2011

(1) Lactic acid bacteria: Lactobacillus plantarum; Lactobacilluscasei; (2) Photosynthetic bacteria: Rhodopseudomonaspalustris; Rhodobactersphaeroides; (3) Yeast: Saccharomyces cerevisiae; Candida utilis; (4) Actinomycetes:Streptomyces albus; Streptomycesgriseus; and (5) Fermenting fungi : Aspergillusoryzae; Mucorhiemalis. The concept of ‘Friendly Microorganisms’ was developed by Japanese horticulturist Teruo Higa, from the University of the Ryukyus in Okinawa Prefecture Okinawa, Japan. He reported that, in the 1970s, a combination of approximately 80 different microorganisms is capable of positively influencing decomposing organic matter such that it reverts into a ‘life promoting’ process. Higa invokes a ‘dominance

principle’ to explain the effects of his ‘Effective Microorganisms’. He claims that three groups of microorganisms exist: ‘positive microorganisms’ (regeneration), ‘negative microorganisms’ (decomposition, degeneration), and ‘opportunist microorganisms’. In every medium (soil, water, air, the human intestine), the ratio of ‘positive’ and ‘negative’ microorganisms is critical, since the opportunist microorganisms follow the trend to regeneration or degeneration. Therefore, Higa believes that, it is possible to positively influence the given medium by supplementing with positive microorganisms.

EM Technology is supposed to maintain sustainable practices such as farming and sustainable living, and also claims to support human health and hygiene, animal husbandry, compost and waste management, disaster clean-up and generally used to promote functions in natural communities. EM has been employed in many agricultural applications .It is also used in the production of several health products and some of it has been used to treat Grey-water, minimize odour from septic tank and remove sludge from drains.

2. Materials and methods

2.1 Experiment tanks and rearing conditions

The experiment was conducted at aquaculture laboratory of Universiti Teknologi MARA Perlis, Malaysia for 28 days. Two aquarium tanks sized 0.909m x 0.458m x 0.450m were prepared and filled with 187 litre of water that are connected to a central bio-filter which the water was circulated with the aid of a submersible pump at a rate equivalent to total tank replacement. The aquaria tanks were covered with thick blue papers to avoid transparency. The tanks were set indoors and water temperature was set to between 28-32oC. Water was aerated on other tanks at least 24 hours for the purpose of removing chlorine and temperature synchronize before poured into the experimental tanks. Water quality parameters were monitored regularly two to three times a week and did not deviate from standard conditions considered favourable for these fishes, i.e. oxygen level of 90-100% saturation; ammonia (measured as NH4

+) do not exceed 0.25 mg/l and nitrite level do not

exceed 0.5 mg/l (Harpaz et al., 2005). Ammonia and nitrite levels were determined using spectrophotometer (HACH DR 2800). During the experimental period, aeration was continuously supplied.

2.2 Fish management

Juvenile Asian seabass which were salt water fish and reared under outdoor condition were transferred to aquaculture laboratory of Universiti Teknologi MARA Perlis, Malaysia. Prior to the beginning of the experiment the juvenile were acclimatized into fresh water conditioned by acclimation rate of -5 ppt day-1 for six days, in the meantime, the fish was treated from salt water diseases. Each tank was stocked to a group of 21 fish populations namely G1 and G2 respectively. After that, 10 samples from each tank were randomly weighed using an electronic balance. Once a week, 10 samples from each tank were randomly weighed using a digital balance to monitor the juvenile growth performance and to update the feeding ration. Stress at weighing was

minimised by systematic operation and there was no feeding on the day of weighing.

2.3 EM preparation

The preparation of EM was based on the recommendation by EM Research Organization Incorporation where a part of non-activated EM can be made into 20 parts of activated EM. Non-activated EM (5%), sugar cane molasses (5%) and treated water (90%) were mixed altogether in a plastic bottle and tightly closed to prevent contamination from other impurities during fermentation process. Water was aerated on other tanks at least 24 hours for the purpose of removing chlorine before the mixing process. Activated EM was ready to use after 4-7 days of fermentation process when the pH drops to 3-3.5, and when it had sweet-sour smell and had changed colour from black to reddish brown. Activated EM was used only within a week after activation is ready because the effective microorganisms are very active and powerful during this period (Higa, 1995). Activated EM was stored in an expandable air-tight container to keep it anaerobic and at room temperature of 20-30oC.

2.4 Experimental diet and design

Feed was in the form of floating extruded pellets, 1mm in diameter, and manufactured for commercial carnivorous fish. For treatment diet, the EM concentration of 0.05ml was mixed to each gram of diet fed to juvenile for 28 days and the experimental diet was stored tightly in plastic containers at room temperature. The experimental diet was ready to use after an overnight from the adding process to ensure EM was well mixed with the pellets. Meanwhile, there was no EM added for the control diet. The juvenile, G1 and G2 were fed control diet and treatment diet respectively, two times daily at 0800-0900

and 1600-1700 hour with a photoperiod of 12 hours light and 12 hours darkness. According to the feeding charts of Asian seabass similar in size to the ones used in the previous study, the feeding rate was around 5.70-7.18% (fish weight range 1.8-11.5g) of the biomass per day. Therefore an initial feeding rate of 5% per day was set and adjusted weekly.

2.5 Statistical analysis

The data were stored and the results are presented as mean±standard deviation. Growth result was analysed using an independent t-test and significant level was set at 5%.

3. Results

The growth and survival results for the experiments are presented in Table 4.1.

Table 4.1 Growth and survival results of Asian seabass juveniles fed treatment supplemented with EM

Treatment Control EMMean body massinitial(g) 13.18±2.70 13.28±4.40Mean body massfinal(g) 32.67±2.90 35.57±4.49SGR (%.day-1) 3.24 3.52Survival (%) 100 100

In term of growth, the SGR for fish fed with treatment diet was 3.52%.day-1

meanwhile fish fed with control diet was 3.24%.day-1. For survival result, both fish fed with control diet and fish fed with treatment diet has 100% of survival as shown in Figure 4.1.

Control EM

3.24 3.52

100 100

SGR Survival

Figure 4.1 Bars chart of SGR (%.day-1) and survival (%) between fish fed with control diet and fish fed with treatment diet of juvenile Asian seabass reared under freshwater closed systems for 28 days.

The mean initial body mass of the experimental fish between fish fed with control diet and fish fed with treatment diet was 13.18±2.70 and 13.28±4.40 g respectively. There was no significant difference (t-test) in the initial body mass between fish fed with control diet and fish fed with treatment diet (t=-0.061; df=18; P=0.952, Appendix C). Meanwhile, results obtained for mean final body mass of the experimental fish were 32.67±2.90 and 35.57±4.49 g for fish fed with control diet and fish fed with treatment diet respectively. Statistical analysis (t-test) indicates that there was no significant difference in the final body

mass between both populations of experimental fish (t=-1.717; df=18; P=0.103, Appendix C).

0 5 10 15 20 25 300

10

20

30

40

ControlEM

Day

Mea

n bo

dy m

ass (

g)

Figure 4.2 Scatter diagram of observed growth performance between fish fed with control diet and fish fed with treatment diet of Asian seabass reared under freshwater closed systems for 28 days.

The Linear Pearson’s Coefficient of Correlations between day and body mass of experimental fish for both experiments indicates a strong correlation with positive slope of 0.9256 for fish fed with control diet and 0.8326 for fish fed with treatment diet as shown in Figure 4.3 and Figure 4.4. For Coefficient of determination, fish fed with control diet was 0.8567 meanwhile fish fed with treatment diet was 0.6932. It was indicates that 85.7% of the variation in body mass of fish fed with control diet was explained by the variation in day and 69.3% for fish fed with treatment diet. Statistical analysis shows sufficient evidence of a linear relationship between day and body mass of experimental fish for both experiments which were P=0 respectively.

Figure 4.3 Regression and correlation graph between day and body mass of juvenile Asian seabass fed with control diet reared under freshwater closed systems for 28 days. Figure 4.4 Regression and correlation graph between day

and body mass of juvenile Asian seabass fed with treatment diet reared under freshwater closed systems for 28 days.

4. Discussion

4.1 Effects of fish sizes on standard error

The mean initial body mass of experimental fish showed a standard deviation of ±2.70 and ±4.40 for fish fed with control diet and fish fed with treatment diet. This indicates that fish sizes for both experiments were uneven. Therefore, it could affect the final body mass of the experimental fish which shows a standard error of ±2.90 and ±4.49 for fish fed with control diet and fish fed with treatment diet respectively. The uneven sizes of experiment fish were due to limited sources of fish and it was a reason on high value in standard deviation for initial and final body mass of experimental fish results. Previous research on protein replacement and growth of juvenile Asian seabass which lupin has been used as protein replacement and indicates a mean initial body mass of 4.21±0.06 g for fish fed with control diet and 4.38±0.01 g for fish fed with treatment diet reared under saltwater condition for 15 days (Katersky et. al., 2007). As a result, the mean final body mass of both experiments fish fed with control diet and fish fed with treatment diet were 11.07±0.12 and

11.75±0.17 g respectively. The research shows a low standard error value for initial body mass of experimental fish, therefore, it would have low value of standard error for final body mass of experimental fish. Due to uneven sizes of the experimental fish, it could affect the experiment results.

4.2 Effects of temperature on growth

Temperature is the most important parameters in affecting growth of Asian seabass where temperature of 31 to 33oC has been studied to be the most optimum temperature to promote growth. Current study has set temperature that range from 28 to 32oC and indicates a SGR of 3.24 and 3.55%.day-1 for fish fed with control diet and fish fed with treatment diet. The SGR result obtained from current study was low compared to the previous studies which using optimum temperature. A study conducted by Katerskyet. al., (2007) on growth of juvenile Asia seabass at different temperature shows the optimal temperature for growth of this species is 31oC. The study shows SGR results of 1.27, 2.32, 6.37, 6.42 and 6.28%.day-1 for temperatures 21, 24, 27, 30 and 33oC respectively. There was significant

difference in the SGR results for temperatures 27, 30 and 33oC which were more than 6 %.day-1 compared to the current study which were around 3%.day-1.

4.3 Effects of diet compositions on growth

Current study sets a diet composition of 43% crude protein, 3% lipid and 6% moisture fed to the Asian seabass for 28 days, the result obtained for SGR was low compared to the previous study. Previous study shows that, Asian seabass has direct significance on the composition diet given. It shows that, the growth of Asian seabass could be effected by the diet compositions.

4.4 Effects of EM on growth of Asian seabass

Current study shows the SGR results for fish fed with treatment diet was 3.52%.day-1, meanwhile fish fed with control diet was 3.24%.day-1. Statistical analysis shows that, there was no significant difference in growth performance for treatment diet compared to control diet. However, the experiment was conducted for 28 days, where the SGR result for fish fed with treatment diet was higher than fish fed with control diet. But, statistical analysis was not supporting the hypothesis that fish fed with treatment diet has growth promoting effects to Asian seabass. The properties of EM are still required further study and research data support. Previous research shows that, EM could improve utilization of apparent metabolisable energy while enhanced feed utilization. The previous studied also has demonstrated and substantiated earlier reports that EM has growth promoting effects and able to reduce serum cholesterol.

4.5 Effects of EM on survival of Asian seabass

In term of survival result, both fish fed with control diet and fish fed with

treatment diet was 100% of survival. It is strongly recommended that further research on survival of Asian seabass for long rearing period be conducted. The properties of EM that are able to reduce serum cholesterol may affect the health and survival of the fish. The survival rate was very high and was not significantly influenced by temperature or size over the duration.

4.6 Effects of rearing days and EM effectiveness on growth

Current study was set it rearing period of 28 days and has strong positive slope of correlation. The Least Square Regression Line of current study were ŷ=0.7096x+12.844 and ŷ =0.843x+13.156 for fish fed with control diet and fish fed with treatment diet respectively where x representing day of rearing meanwhile ŷ representing body mass of fish. Therefore, it is indicated that, for every addition increase in day, body mass of fish is expected to increase by an average of 0.7096 g for fish fed with control diet and 0.843 g for fish fed with treatment diet. It is indicated that, the variable of day in this experiment has different effects on growth of Asian seabass in which, fish fed with treatment diet could have higher body mass compare to fish fed with control diet for longer period of rearing. However, an experiment should be conducted to prove this hypothesis in future. In term of EM effectiveness, there was no particular research to determine on EM effectiveness or EM concentration required to correlated with rearing period. Most studies shows that, EM could improve water quality and fish health in short period but it was ambiguous evidence on EM could promoting growth of fish as results obtained from the current study. Current study has revealed the hypothesis that EM might be effective if rearing days is prolonged.

4.7 Effects of EM on water quality

The amount of solid waste (output waste) produced by the Asian seabass was significantly different between fish fed with control diet and fish fed with treatment diet which effects water quality. During the experiment, the solid waste from fish fed with treatment diet was in small quantity and ash’s type compared to the fish fed with control diet. This is due

to the characteristics of EM itself as an organic decomposer. In order to maintain water quality during the experiment, the water quality had been set and ensure its not deviate from standard conditions considered favorable for these fishes, i.e. oxygen level of 90-100% saturation; ammonia (measured as NH4

+) do not exceed 0.25 mg/l and nitrite level do not exceed 0.5 mg/l.

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