European International Journal of Science and Technology Vol. 4 No. 5 June, 2015

THE EFFECTIVENESS OF FISH SILAGE AS ORGANIC FERTILIZER ON POST-HARVEST QUALITY OF PAK CHOY (Brassica rapaL. subsp. chinensis)

Nurul Ulfah Karim1,3*, Mohd Farid Mohd Ali Lee1, Adzemi Mat Arshad2

1School of Fisheries and Aquaculture Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, MALAYSIA

2School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, MALAYSIA

3Institute of Aquaculture Tropical, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, MALAYSIA

*Corresponding author: [email protected]

ABSTRACT A study was conducted to determine the physiochemical of liquid fish silage (LFS); pH and macronutrient content made by fish waste and to reveal the effectiveness of LFS toward the growth, yield, pigment content and post-harvest quality of pakchoy. LFS treatments were prepared at five different concentrations; 1.0, 2.5, 5.0, 7.5 and 10.0%. Each treatments were compared with plant fertilized with commercial fertilizer (N-P-K 15:15:15) using recommended nutrient per hectare. After 14 days of fermentation, the pH values of LFS were stable at 4.5. LFS contains 1.84% N, 0.50% P, 0.41% K, 0.36% Mg and 0.84% Ca. The results showed that LFS at 5.0, 7.5 and 10.0% produced similar plant growth, yield, pigment content and post-harvest quality as plant fertilized by commercial fertilizer. In addition, this study recommend to use 5.0% LFS as it more economical compared to 7.5 and 10.0%.

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INTRODUCTION The demand for fish increased with increasing human populations. However, it has been estimated that for each tons of fish eaten, an equal volume of fish material were discarded either as waste or as a low value by- product (Anon, 2003). There are some potential for gaining more value from fish wastes. Fish rich in valuable minerals, enzymes, pigments and flavors that are required by many industries including food, agriculture, aquaculture and pharmaceuticals (Archer et al., 2001). Elsewhere, fish waste was converted into fishmeal or utilized in the production of organic fertilizers and composts, which have significant benefits over chemical-based products (Archer et al., 2001). Fish fertilizer products such as fish hydrolysates, fish emulsion, fish soluble nutrient and fish silage have widely being used in crops productions and was reported produced similar yields as conventional fertilizer (Wyatt &McGourty, 1990). Silage production is considered the best method to address the problem of environmental production due to processing waste and underutilized by-catch. Fish silage process has been reported to be a feasible, simple and lower-cost alternative and no skilled personnel is needed (Vidottiet al., 2003). Besides, the capital investment is significantly less compared to other fish fertilizer product in market. Fish silage contain more valuables nutrient that is good for plant growth and soil microbes. Pak choy belonging to family Brassicaceace is becoming increasingly popular with gardeners as an alternative to cabbage (Royal Horticulture Society [RHS], 2010). The aim of this study is to evaluate the productions of fish silage as one of the alternative ways in managing fish wastes and by-products by converting these wastes into liquid organic fertilizer. The effectiveness of this product were compared to the commercial fertilizer used on pakchoy cultivation.

MATERIALS AND METHODS

3.1 Experiment preparation 3.1.1 Liquid fish silage (LFS) preparation Liquid fish silage (LFS) of tread finseabream, Nemipterusjaponicus from surimi processing waste (non-edible parts) were prepared according to Fagbenro&Jauncey (1995). Four kilograms of emulsified fish waste were mixed with 750 g of molasses and 250 g of Lactobacillus acidophilus starter culture (for lactic acid anaerobic fermentation). The mixture were fermented until reached pH 4.5 and filtered thoroughly 1mm mesh screen.

3.1.2 Seeds sowing Seeds of Brassica rapaL. subsp. Chinensis with uniform size were treated with fungicides and sown in germination tray. Twelve days old seedlings were transplanted into 3000 cm2 plot and watered daily.

3.1.3 Fertilizer applications The fertilizers were prepared by diluting concentrated LFS to five different concentrations; 1.0, 2.5, 5.0, 7.5 and 10.0% in 100 ml water. Fertilizers were applied at interval 5 days for 30 days. Seven treatments were studies in this experiment for evaluating the efficacy of LFS (Table 1). All treatments were replicates 3 times.

164 European International Journal of Science and Technology Vol. 4 No. 5 June, 2015

Table 1: Seven different treatments used to evaluate the efficacy of LFS Treatment Concentration (%) 1 Commercial (NPK 15:15:15) 2 Control (water) 3 1.0% LFS in 100 ml water 4 2.5% LFS in 100 ml water 5 5.0% LFS in 100 ml water 6 7.5% LFS in 100 ml water 7 10.0% LFS in 100 ml water

3.2. Physiochemical of liquid fish silage (LFS) 3.2.1 Determination of pH The pH values of LFS were measured at interval of 7 days within 70 days of storage. pH values were measured by immersing electrode in the supernatant solution of 1:2 volume.

3.2.2 Determination of macronutrient composition Nitrogen content were determined using Kjeldahl method (KjeltecTM 2100, Denmark) according method by AOAC (1990). The macro elements; phosphorus, potassium, magnesium and calcium were determined using atomic absorption spectrophotometry [AAS] (AAnalyst™ 200, USA)according to method by Peter et al., (2003).

3.3 Analysis on the growth parameter of pakchoy Height of pakchoy was measured from plant base to the highest tip of leafat interval of 5 day within day 30 of transplant. Numbers of leaves growing on plant were recorded every 5 days until the 30th day. Areas of the leaf were analyzed using Image J software 1.74v (National Institute of Health, USA). For total yield and mean fresh weight, pakchoy were weighed immediately once harvested to determine the total yield production per plot and mean fresh weight.

3.4 Analysis on the pigment content of pakchoy Chlorophylla and b were measured using UV spectroscopy (UV Mini 1240, US) according tomethod by Arnon (1949). Leaf were cut into small pieces with the weight about 100±0.1 mg. Samples were homogenate and 10 ml of 80 % acetone (acetone: water 80:20 v: v) were added. The homogenate were filtered and the filtrate were collected in a testtube. Wavelength for chlorophyll a and b were set at 663 nm and 645 nm on spectrophotometer.

Calculations: Use Arnon's equation (below) to convert absorbance measurements to mg Chl g-1 leaf tissue.

Chlorophyll a (mg g-1) = [(12.7 × A663) - (2.6 × A645)] × acetone (ml) Leaf tissue (mg) Chlorophyll b (mg g-1) = [(22.9 × A645) - (4.68 × A663)] × acetone (ml) Leaf tissue (mg) Total Chlorophyll = Chlorophyll a + Chlorophyll b.

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3.5 Analysis on post-harvest quality of pakchoy Hardness and crispness were analyzed using texture analyzer (TA.XT2, UK). The leaf colors of pakchoy were measured using colorimeter (Konica Minolta CR-400, Japan).Fifth marketable leaf from each plant was selected for analysis(Tshikalange& Van Averbeke, 2006). Measurement of L*, a* and b* value were taken at the center of the 5th leaf.

3.6 Statistical analysis All measurements were performed in triplicate per treatment and the values were express as the mean ±standard deviation. The collected data were analyzed using one way ANOVA and hypothesis test were analyzed using Tukey Test at significance level 0.05. RESULTS AND DISCUSSIONS

4.1 Physiochemical of LFS 4.1.1 pH value of LFS pH value of LFS were recorded at range between 4.40±0.00 and 6.53±0.06 during 70 days fermentation and storage period (Table 2). The highest pH value was recorded at day 0 and the lowest were recorded at day 21. In this study, pH decreased from 6.53±0.06 to 4.50±0.06 within 10 days of fermentation after inoculation of LAB(Table 2).

Table 2 pH value of LFS during 70 days fermentation and storage Condition Days pH 0 6.53±0.06a Fermentation 7 4.63±0.06b (1st fertilizer application) 14 4.50±0.00bc 21 4.40±0.00c 28 4.47±0.06c 35 4.43±0.06c Storage 42 4.43±0.06c 49 4.43±0.06c 56 4.43±0.06c 63 4.47±0.06c 70 4.47±0.06c Values are mean±stdev. n = 3. Superscripts a,b,c show the significant difference, (p<0.05).

4.1.2 Macronutrient composition LFS have a low macronutrient but rich in trace minerals. In this study, LFS contain 1.84±0.38% nitrogen (N), 0.50±0.09% phosphorus (P), 0.41±0.05% potassium (K), 0.36±0.02% magnesium (Mg) and 0.84±0.12% calcium (Ca) (Table 3). This were similar as reported by Blatt& Sanford (1990) which contain 1–3% of nitrogen, 0.4–2.6% phosphorus and less than 1% for potassium, calcium and magnesium. Tatterson& Windsor (2006) stated the composition of fish silage were similar to the material from which it prepared. According to Palaniet al., (2014) nitrogen, potassium and calcium content in Nemipterusjaponicus are 2.91 % N, 0.26% K and 0.79% Ca, respectively.

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Table 3 Nutrient composition and percentage of LFS made from threadfin seabream waste

Macronutrient Composition (%) Nitrogen 1.84±0.38 Phosphorus 0.50±0.09 Potassium 0.41±0.05 Magnesium 0.36±0.02 Calcium 0.84±0.12

4.2 Effect of LFS on growth of pakchoy Plant treated at 10.0% LFS produced highest height; 27.24±1.49 cm while the lowest height are the controls which is 14.78±0.90 cm after 30 days of cultivation (Table 4). The height of pakchoy treated at 5.0, 7.5 and 10.0% LFS were not significantly different (p>0.05) to plant received commercial fertilizer after 30 days cultivation. In the other hand, controls and plants treated at 1.0 and 2.5% LFS showed significantly (p<0.05) shorter compared to plant received commercial fertilizer (Figure 1).Plant received commercial fertilizer produced highest mean leaf number, which is 13.25±0.66 while the lowest mean leaf number obtained in controls at 8.17±0.52 after 30 days cultivation (Table 5). No significant difference (p>0.05) in mean leaf number were observed between plants received commercial fertilizer and plants received 5.0, 7.5 and 10.0% LFS(Table 5). Leaf areas were determined once plants were harvested after 30 days cultivation. Among the treatments, plant received 5.0% LFS produced largest mean leaf area, which is 1174±39 cm2 while the smallest mean leaf area were obtained in controls at 251±32 cm2 (Figure 2). Pak choy received 2.5, 5.0, 7.5 and 10.0% LFS produce similar leaf area (p>0.05) compared to plant received commercial fertilizer. However, plants in controls and received 1.0% LFS produce smaller leaf area (p<0.05) compared to plants received commercial fertilizer (Figure 2).

30.00

25.00

20.00

15.00

Mean plant height (cm) height plantMean 10.00

5.00

0.00 0 5 10 15 20 25 30 Cultivation (days) Figure 1 Mean plant height of pakchoy during 30 days cultivation in different treatments. n = 3. ö NPK; +Control; Ø 1.0% LFS;
2.5% LFS; r 5.0% LFS; œ 7.5% LFS; ¤ 10.0% LFS

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1600.00 a 1400.00 ac a ac 1200.00

1000.00 c 800.00 bc 600.00

Mean leaf area (cm²) area leaf Mean 400.00 b 200.00

0.00 NPK Control 1.0% 2.5% 5.0% 7.5% 10.0% LFS LFS LFS LFS LFS Treatment

Figure 2 Mean leaf area of pakchoy after 30 days cultivation in different treatments. n = 3. Different small letters on top of the bars indicate significant differences (p<0.05)

Yoshizawaet al., (1981) reported the application rate of 120 kg N ha-1 found to be optimal for pakchoy growth. Tshikalange&Averbeke(2006) added that pakchoy growth tended to peak when nitrogen were at the rate of 200 kg N ha-1 but decline when the application rate exceeded 200 kg N ha-1. Therefore, low nitrogen application rate is where the nitrogen is less than 120 kg .ha-1, medium or recommended rate nitrogen at range of 120 kg-200 kg ha-1. Meanwhile high application rate is when nitrogen content exceed 200 kg. ha-1. In this studies, pakchoy treated at medium and high application rate of LFS produced similar growth as plant fertilized with commercial fertilizer because its provide suitable nutrient requirement (Table 6). Rachman&Suwars (1990) reported that application of nitrogen increases leaf dimensions (length and width of leaf) that are may leads to yield rise. Castelliet al., (1990) added that increasing the amount of nitrogen increases the number of leaves, plant height, stem diameter and wet leaf weight. Meanwhile, Li- zhilinet al., (1997) stated that plant height is increased significantly due to nitrogen application.

Plants treated with 7.5% LFS produced highest total yield; 1587 g with mean fresh weight 132.21±21.16 g. While the lowest yield obtained in controls; 129 g with mean fresh weight 10.73±1.34 g (Table 7). No significant different (p>0.05) of mean weight between pakchoy treated with 5.0, 7.5 and 10.0% compared to pakchoy treated with commercial fertilizer. They are capable to produce between 10-12 times yields than the controls (Table 7). Treatment of 5.0, 7.5 and 10.0% provide suitable nutrient requirement for the growth of pakchoy. Blatt (1991) stated that the applications of same nitrogen rate of fish silage produce a comparable yield to broccoli, cauliflower and cabbage cultivation to chemical fertilizer in most seasons. Irshad&Javed (2006) added high yield were obtained for mung bean and okra cultivation when using fish as fertilizer compared to Nitrogen-Phosphorous-Potassium (NPK) and urea fertilizer.

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Table 4 Mean height of pakchoy during 30 days cultivation in different treatments

Plant height (cm) Treatments Day 0 Day 5 Day 10 Day 15 Day 20 Day 25 Day 30 Commercial 4.88±0.25a 8.71±0.32a 13.65±0.40abc 18.33±0.75a 21.92±1.02a 24.17±0.94ad 25.75±1.60a (NPK) Control (water) 5.54±0.82 a 9.45±0.85a 12.33±0.87b 14.16±0.63b 14.22±0.55b 14.76±0.96b 14.78±0.90b 1.0% LFS 5.38±0.62 a 9.07±0.88a 12.74±0.61bc 15.43±0.82b 18.39±0.70c 19.92±0.69c 19.62±0.68c 2.5% LFS 5.62±0.06 a 9.94±0.12a 14.98±0.50a 18.79±0.73a 21.87±0.40a 22.66±0.68d 22.13±0.82c 5.0% LFS 5.17±0.54 a 9.32±0.42a 14.28±1.15ac 19.17±0.64a 23.67±0.56a 26.31±0.78a 27.06±0.72a 7.5% LFS 6.01±0.31 a 9.98±0.55a 14.40±0.60ac 19.33±1.13a 23.17±0.72a 24.88±1.00ad 27.05±0.91a 10.0% LFS 5.85±0.40 a 10.11±0.23a 13.97±0.21abc 19.00±0.38a 22.07±1.91a 25.44±0.99a 27.24±1.49a Values are mean±stdev. n = 3. Different superscripts a,b,c in same column indicated significant difference (p<0.05).

Table 5 Mean leaf number of pakchoy during 30 days cultivation in different treatments

Leaf number Treatments Day 0 Day 5 Day 10 Day 15 Day 20 Day 25 Day 30 Commercial 2.08±0.14a 3.58±0.52a 5.33±0.29a 7.33±0.52a 9.33±0.72a 11.92±0.38a 13.25±0.66a (NPK) Control (water) 2.00±0.00a 3.67±0.14a 4.92±0.14a 5.75±0.25b 6.50±0.43b 7.42±0.38b 8.17±0.52b 1.0% LFS 2.00±0.00a 3.75±0.00a 4.92±0.14a 6.33±0.29bc 7.58±0.14bc 9.00±0.43cd 9.75±0.66c 2.5% LFS 2.08±0.14a 4.00±0.00a 5.42±0.29a 7.25±0.25ac 8.83±0.52ac 10.17±0.14d 11.08±0.38cd 5.0% LFS 2.08±0.14a 3.75±0.25a 5.42±0.38a 7.50±0.25a 9.33±0.14a 10.33±0.58d 11.92±0.38ad 7.5% LFS 2.08±0.14a 4.08±0.14a 5.58±0.14a 7.58±0.38a 8.92±0.63a 10.17±0.38d 11.83±0.63ad 10.0% LFS 2.00±0.00a 3.89±0.19a 5.25±0.43a 7.33±0.38a 9.06±0.34a 10.33±0.29d 11.78±0.48ad Values are mean±stdev. n = 3. Different superscripts a,b,c in same column indicated significant difference (p<0.05).

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Table 6 Volume of concentrated LFS used during 30 days pakchoy cultivation and their N-P-K contents that were sorted based on their nitrogen contents. Total Total Total Total Amount of concentrated LFS nitrogen phosphorus potassium Treatments nutrient used per plot/12 applied/ applied/ plot applied/ plot applied/ha (kg) plants (ml) plot (g) (g) (g) Control (water) - Trace Trace Trace Trace 1.0% LFS 48 1.06 0.29 0.25 35 – 10 – 8 2.5% LFS 120 2.64 0.72 0.62 88 – 24 – 21 5.0% LFS 240 5.29 1.43 1.24 176 – 48 – 41 Commercial - 6 6 6 200– 200 – 200 (NPK) 7.5% LFS 360 7.93 2.16 1.85 264 – 72 – 62 10.0% LFS 480 10.58 2.87 2.47 353 – 96 – 82 *Plot contain 3 replicates and each replicate contain 4 plants with planting area 250 cm2/plant or 3000 cm2/12 plants **Total concentrated LFS is volume LFS used for 4 times fertilizer application (7 days interval) during 30 days cultivation

Table 7 Mean fresh weight and total fresh yield of harvested pakchoy after 30 days cultivation in different treatments. Treatments Mean fresh weigh (g) Total yield/plot (g) Commercial (NPK) 112.67±3.02a 1352 Control (water) 10.73±1.34b 129 1.0% LFS 32.82±0.52b 394 2.5% LFS 65.33±5.51c 784 5.0% LFS 110.12±6.77a 1321 7.5% LFS 132.21±21.16a 1587 10.0% LFS 112.58±7.18a 1351 Values are mean±stdev. n = 3. Different superscripts a,b,c indicated significant difference (p<0.05).

4.3 Effect of LFS on pigment content of pakchoy leaf The highest chlorophyll a obtained in pakchoy treated with 10.0% LFS while lowest chlorophyll a obtained in controls (Table 8). The highest values of chlorophyll b in plant were treated with controls, meanwhile lowest value of chlorophyll b obtained in plant received 2.5% LFS (Table 8). The highest value of total chlorophyll were recorded in plant received 10.0% LFS while, lowest value were obtained in controls (Table 8). Dikshit&Paliwal, (1989) reported that application of high nitrogen directly increased the chlorophyll content and leaf surface area. In this studies, high chlorophyll content were observed in plant treated with high nitrogen treatment; 5.0, 7.5, 10.0% LFS and commercial. In the other hand, pakchoy in controls, 1.0 and 2.5% LFS, plant produce lower chlorophyll content compared to others, but the values are small and show no significant different between them (p>0.05).

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Table 8 Chlorophyll a, b and total chlorophyll content in 100 mg fresh pakchoy 5th leaf in different treatments Treatments Chla (mg) Chlb (mg) Total Chl (mg) Commercial (NPK) 1.257±0.08a 0.344±0.06a 1.602 Control (water) 0.986±0.10a 0.466±0.16a 1.452 1.0% LFS 1.150±0.14a 0.356±0.07a 1.507 2.5% LFS 1.193±0.10a 0.321±0.07a 1.514 5.0% LFS 1.274±0.16a 0.326±0.15a 1.600 7.5% LFS 1.223±0.17a 0.371±0.20a 1.594 10.0% LFS 1.161±0.15a 0.449±0.04a 1.610 Values are mean±stdev. n = 3. Different superscripts a,b,c in same column indicated significant difference (p<0.05)

4.4 Effect of LFS on post-harvest quality of pakchoy Pak choytreated with7.5% LFS had the lowest value of both hardness and crispness at 0.48±0.01 forces kg-1 and 2.40±0.09 forces kg-1, respectively. Meanwhile, the highest value obtained in controls to give amount of 1.44±0.20 forces kg-1 and 4.51±0.32 forces kg-1, respectively (Figure 3).The hardness and crispness of pakchoy petiole that received 2.5, 5.0, 7.5 and 10.0% LFS were similar (p>0.05) compared to petiole of pakchoy treated with commercial fertilizer (Figure 3). In the other hand, plant significantly produced higher value of hardness and crispness (p<0.05) when treated in controls and 1.0% LFS compared to others treatments

5.00 b

4.50 c 4.00

3.50 a a 3.00 a a a 2.50

2.00 b Force (kg) Force 1.50 c 1.00 a a a a a 0.50 0.00 NPK Control 1.0% 2.5% 5.0% 7.5% 10.0% LFS LFS LFS LFS LFS Treatment

Crispness Hardness

Figure 3 Hardness and crispness of 5ft petiole harvested pakchoy after 30 days cultivation in different treatments. n = 3. Different small letters on top of the bars indicate significant differences between parameter (p<0.05)

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For value of a* showed an increasing of greenness plant treated with commercial fertilizer (- 15.64±1.53) while plant treated with 1.0% LFS showed the lowest value of greenness (-13.71±0.65) (Table 9). For value of b*, plant received 1.0% LFS showed the most less of yellowness (15.89±1.03) compared to plant treated with commercial fertilizer (18.64±2.37) (Table 9).Green color of leaf depends on concentration chlorophyll present in leaf while the concentration of chlorophyll are affected by amount of nutrient supplied to the plants especially nitrogen (Taiz&Zeiger, 2012). However, Table 8 showed no significant different of pigments and thus a similar result were shown in colour of pakchoy(Table 9).

An observation found that plants treated with high nitrogen rate (NPK, 5.0, 7.5 and 10.0%) produced darker leaf (low L* value) and more green color (low – a* value) compared to plant received lower nitrogen rate (controls, 1.0 and 2.5%) (Table 9). Lincoln & Eduardo (2012) stated that deficiency of nitrogen leads to loss green color in the leaves, decrease leaf area and intensity of photosynthesis.Comparison among all treatments to commercial fertilizer on growth, yield, pigment and post-harvest quality of pakchoy were showed in Table 10. Based on the results, it shows that treatment that received 5.0, 7.5 and 10.0% LFS produce similar growth, yield, pigment content and post-harvest quality compared to plant treated with commercial fertilizer using recommended nutrient per hectare.

Table 9 Value of L*, a and b* in 5th harvested pakchoy leaf after 30 days cultivation in different treatments Treatments L* a* b* Commercial (NPK) 37.84±1.92a -15.64±1.53a 18.24±2.77a Control (water) 39.28±0.38a -13.89±0.85a 15.93±1.43a 1.0% LFS 39.03±1.65a -13.71±0.45a 15.89±1.03a 2.5% LFS 39.82±0.48a -13.79±1.23a 16.87±1.40a 5.0% LFS 38.92±2.46a -15.51±1.42a 18.64±2.37a 7.5% LFS 37.23±2.73a -14.47±2.33a 17.80±3.60a 10.0% LFS 37.47±0.92a -14.51±0.72a 17.30±1.54a Values are mean±stdev. n = 3. Different superscripts a,b,c in same column indicated significant difference (p<0.05)

Table 10 Comparison between all treatments used in this study with treatment fertilized with commercial fertilizer on growth, yield, pigment and post-harvest quality of pakchoy Plant growth Yield Post-harvest quality Pigment Treatments Plant Leaf Leaf Fresh Leaf Chl Hardness Crispness Chla Height number area weight color b Control * * * * * * n.s n.s n.s (water) 1.0% LFS * * * * * * n.s n.s n.s 2.5% LFS * * n.s * n.s n.s n.s n.s n.s 5.0% LFS n.s n.s n.s n.s n.s n.s n.s n.s n.s 7.5% LFS n.s n.s n.s n.s n.s n.s n.s n.s n.s 10.0% LFS n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s = no significant differences (p>0.05) with plant fertilized with commercial fertilizer * = significant differences (p<0.05) with plant fertilized with commercial fertilizer

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CONCLUSION LFS has a great potential to be used as organic fertilizer for crops production. LFS at 5.0-10.0% concentration is very effective for growing pakchoy as it would produce same yields and quality when treat with commercial fertilizer.

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