Type and fertiliser used in N:P:K ratio for mass culture of marine
1. Introduction
Sustainability in the aquaculture industry depends on several factors including the minimum production cost in comparison to the yield, unexpected environmental conditions which affect the farm and practices in the farm management itself. These factors are inter-connected and always incur a synergistic effect on the issue of sustainability. Live feeds as the fundamental needs for larval rearing and fry production have to be prioritised for sustainable farming activity. Dependency on imported sources of live feeds or inert feed will increase the production cost. Thus, the continued activity of screening, stocking and maintaining some local species as an option for live feed production is economically necessary.
Live feeds are an important basic diet for newly-hatched fish and shrimp larvae as they still have an incomplete digestive system and are lacking in enzymes. They are still at a very young stage to generate their own required nutrients or convert them from any pre-cursor obtained from a diet. They need a ready-made diet with readily available nutrient to be absorbed through their digestive system. There have been many species suggested or tested for their potential as live feed. All test animals were mostly zooplankton in nature and must meet the requirement as live feed. They must be in a compatible size with the mouth size or gape of the larvae predator, or they cannot be swallowed. Since larvae are still weak to track down the food, the wave created by the prey will be a great help, thus ‘active’ swimming prey is preferred. The most important role of a prey is the ability to supply energy and other nutrients which are essential for the larval survival and growth. Live feeds, as the starter diet in larval rearing and fry production must be continuous in supply. Good, nutritious and compatible-size prey must be able to reproduce fast to meet the requirement and adaptable to a simple mass-production technique.
1.1. Copepods as live feeds
The conventional live feed, brine shrimp and rotifers, are considered unsuitable as live feed if compared to copepods in term of nutritional value.
Despite these positive findings, rotifers and
Another copepod group, a Cyclopoida,
1.2. Microalgae
Microalgae are a diverse group of unicellular autotrophs inhabiting almost all aquatic water bodies. Microalgae are rich in many specific and attractive compounds [22] and their nutritional values for aquaculture had been highlighted [23]. Production of microalgae is mandatory in the hatchery as it is a basic and nutritious diet for live feed, specifically the zooplankton. However, its mass production is generally costly due to huge manpower, space requirements and operation which usually related to the cost of the energy used. A good strategy in manipulating the culture environment, particularly during the production process of microalgae would scale down the operational cost.
Light plays a fundamental role in the development of microalgae through photosynthesis. It is one of the major environmental factors which control the performance of microalgae phototrophic growth and productivity [24, 25, 26]. Light may either be natural or supplied by fluorescent tubes giving the maximum effective radiation which can be absorbed by the pigments of the microalgae. Light intensity plays a vital role, but the requirements vary with the culture depth or volume as well as the density of the algae in the culture. At a higher volume, light intensity must be increased to enable it to penetrate through the culture. However, an extreme light intensity may result in photo-inhibition which reduces the photosynthetic rates and growth [27,28]. Furthermore, overheating due to artificial or natural illumination should be avoided in microalgal culture. The most often employed light intensity is 1000 lux which is suitable for Erlenmeyer flasks but 5000-10000 lux is needed for a greater volume of microalgal culture [29, 30]. The duration of illumination can be varied where photosynthesis of microalgae can be enhanced or increased in the light/dark (LD) cycle (usually 12:12 or 14:10 LD, maximum 16:8 LD). For some microalgae, photosynthesis rate could also be increased exponentially with increasing light/dark frequencies where a longer period of dark in relation to the light period can further increase photosynthetic efficiencies but not vice versa [31]. The illuminations also affect the nutrient utilisation in the culture vessel [32].
A cost-effective and nutritionally-adequate alternative to costly maintenance of live microalgae is the production of moist-microalgae concentrates. It is seen to simplify hatchery procedures and has shown promising potential in the aquaculture industry [33,34].The storing of microalgae concentrates in moist form under low temperature can preserve their high nutrient composition and excellent cell viability [35,33]. Juvenile pacific oyster (
Preparation of concentrated condition of microalgae usually involves centrifugation technique. Nonetheless, although this technique has been successfully applied and utilised for preparing microalgae concentrates, it poses some limitations. First, the process involves exposure of cells to high gravitational and shear forces deteriorating the cell structure with the leaking of nutritional contents. Second, centrifuging large volumes of cultures is time-consuming and requires expensive equipments. Several alternative procedures, less damaging to the cells, which can be applied are filtration [40], foam fractionation [41] and flocculation [33, 34, 38]. Previous studies have observed the excellence of ultrafiltration technique in preserving and retaining the cellular structure and properties of fragile algal cells with little loss of material [42, 43].
The level of natural resources exploitation for aquaculture purposes is commonly high. Coastal land and mangroves forests always become the target area for brackish-water aquaculture ponds. The water source of this area, which is always from the nearby river estuary and lagoon, is also used as the live feeds (zooplankton and microalgae) source. Nonetheless, the supply is always seasonal and could become unavailable unexpectedly due to many factors and natural phenomena. This chapter aims to discuss the possible ways to produce local live feeds, a marine microalgae species and a planktonic copepod, sustainably using a simple technique for larval-rearing purposes. Maintaining local species is hypothesised to be more economical and practical. The usage of the microalgae as an enrichment element for live feed copepods will be proved.
2. Methodology
Seawater samples were obtained from Bidong Island, Terengganu. The collection was made by lowering a Niskin water sampler to a required depth, following the light-penetration depth. Concentrated water samples were then transferred into chilled, white-plastic containers and brought back to the laboratory for microalgae isolation process. Successive plating out on agar plates was performed in order to select the desired marine
The microalgae was then cultured for the preparation of moist concentrates in the temperature controlled room (20±2°C) using the standard batch culture method. Triplicate of actively-growing starter cultures were inoculated into 30 litres acrylic tanks enriched with Conway medium under constant illumination (cool-white type; 110 watts). All cultures were started with an initial inoculum of 2x106 cells mL-1. Cultures were aerated continuously using humidified filtered air. Evaporation in the culturing tanks was kept to a minimum by covering the top of the tanks. Cellular density of microalgae cultures was monitored daily using a Neubauer haemocytometer [29]. Scanning electron microscopic observation was also done to determine the ultra structure of the cell. Measurement of radius and height of the target microalgae cells was done under the advanced research microscope (Model Nikon Eclipse 80-i, Japan) and twenty individual cells were measured for the calculation of cell biovolume to avoid biasing results. Cell biovolume was calculated as assumed round-shape volume with the following formula proposed by Sun and Liu [44]:
Where, π= 3.142, R= radius of cell
Specific growth rate was calculated from the expression as proposed [45] which is shown below:
Where, µ = specific growth rate, F1= biomass at time harvest, t1 and F0= biomass at time zero, t0.
Doubling time was computed based on the formula as proposed [45] which is shown below:
Where, T = doubling time, µ = specific growth rate.
All microalgae cultures were grown to late-logarithmic phase for the preparation of concentrates via ultrafiltration technique. The concentrated aqueous suspensions of microalgae were filtered through a membrane filter (0.1µm pore size) to remove access water from the suspension without rupturing the microalgae, thereby obtaining the microalgae concentrate or paste. Cell viabilities of microalgae concentrates were assessed using Eosin dye as a viability assay on the basis of its penetration into non viable-cells based on the expression as proposed [46]:
The harvesting efficiency or percentage recovery (%) was evaluated by comparing the remaining total number of cells in the concentrate with the total number of cells before filtration with the following expression:
Where, CB = total number of cells before filtration, CA = total number of cells after filtration
Microalgal concentrates were compared to live cultures of the same algae as food for marine copepods. Copepods were obtained from existing culture in UMT’s laboratory. Two different sets of cultures were done using a Petri dish where each of them was fed with live and microalgae concentrate respectively. Individual copepods were counted daily under the Leica stereo microscope before being fed (1 drop). The maximum specific growth rate (K) was calculated [47] as shown below:
Where, K = specific growth rate, X1= the number of copepods at harvest time, t1 and X2= the number of copepods at time zero, t0
The doubling time was computed as:
Where, т = doubling time, K = specific growth rate.
Pure strains of
Microalgae were grown in autotrophic conditions as a monospecific axenic culture in different volumes (250mL, 500mL and 2000mL) containing Conway media. 25mL of pure strain with the cell density of ~2 x 106 cells mL-1 were transferred to each Erlenmeyer culture flask and kept at complete illumination provided by luminescent tubes (1000 Lux). Carbon source was provided by bubbling sterile 2% (v/v) CO2 in air through the cultures. Culture flasks were maintained at a constant temperature (22
Mean cell count and specific growth rate were calculated using the formula
An investigation was made to see the adaptability of the local marine
Culture containers were well-cleaned with bleach and rinsed thoroughly before filling up with 1L of the farm water (salinity of between 20-25ppt). The marine
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15 | 15 | 15 | 14.7 | 14.7 | 14.7 |
8 | 8 | 2 | 7.84 | 7.84 | 1.96 |
16 | 8 | 6 | 15.68 | 7.84 | 5.88 |
12 | 6 | 4 | 11.76 | 5.88 | 3.92 |
12 | 8 | 4 | 11.76 | 7.84 | 3.92 |
Sampling of microalgae cells was done daily and counting was carried out using a Neubauer Hemocytometer covered with glass slide under a compound microscope.
The growth rate, divisions per day, and generation time or doubling time was calculated following [49]
Where, No and Nt = final and initial populations at time t1 and time t2, respectively.
Since sample was collected daily, therefore, t2 – t1 = 1.
Detailed observation on the reproduction performance of a zooplankton depending solely on a
The investigation on the reproduction performance started with fifteen gravid females of
The population growth of
Where, t is the culture days, No and Nt is the number of copepods at the initial and final selected time interval. The doubling time (Dt) was calculated by dividing loge2 by the population growth rate (K) of all stages of
Although cyclopoid copepods are known to suspend in water column,
3. Result and discussions
The ultra-structure of the
The cell has an average cell biovolume of 5.26±0.87 µm3. The cell densities changed following the culture period in both culture of concentrates (paste) and live condition (although they both started at the same density). Nonetheless, they followed more or less the same growth patten. The variation in cell densities during the experimental period is shown in Figure 2. Cell density of
Exponential (log) phase (days 0-6),
Declining of relative growth rate phase (days 6-12),
Stationary phase (days 12-22),
Death phase (days 22-26).
Copepod species,
The ultrafiltration technique which was used to concentrate the
The use of
It is very important for hatcheries to be able to maintain the stock for microalgae for their sustainable live-feeds supply. Batch cultures need to be maintained under optimal environmental conditions and in a suitable culture vessel which will not affect the cell density and quality. Comparison on the effect of photoperiod and culture sizes between
It is well-documented that, in natural conditions, microalgae growth is not curtailed by ambient environmental conditions because the growth rate is just enough for species survival. However, their multiplication rate is highly influenced by various environmental parameters. In an
Highest cell density and specific growth rate were recorded in selected species cultured in 250mL culture flask compared to the cultures in 500mL and 2000mL flasks (Table 2). The highest cell density was achieved during the end of the log phase. Cell density of early stationary phase, which is the end of the log phase for
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250 | Day 10 | 112.5±2.36a | 0.52±0.01a |
500 | Day 11 | 110.17±1.77a | 0.46±0.04b | |
2000 | Day 13 | 92.2± 0.87a | 0.34±0.01c* (ac) | |
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250 | Day 10 | 12.460±0.018a | 0.203±0.002a |
500 | Day 12 | 10.889±0.013b** (ab) | 0.145±0.001b* (ab) | |
2000 | Day 14 | 8.225±0.001c** (ac & bc) | 0.037±0.003c** (ac), * (bc) | |
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250 | Day 10 | 117.53± 0.84a | 0.4749±0.0007a |
500 | Day 11 | 91.0± 0.55a | 0.4081±0.0002b | |
2000 | Day 14 | 86.13±0.81a | 0.3166±0.0007c* (ac) |
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24:0 | Day 13 | 112.5±2.36a | 0.34±0.01a |
12:12 | Day 17 | 110.17±1.77a | 0.25±0.01b | |
8:16 | Day 23 | 92.2± 0.87a | 0.19±0.02c* (ac) | |
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24:0 | Day 12 | 8.225±0.001a | 0.129±0.003a |
12:12 | Day 17 | 5.293±0.009b** (ab) | 0.061±0.002b* (ab) | |
8:16 | Day 22 | 4.453 |
0.037±0.003c** (ac) | |
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24:0 | Day 14 | 86.60 ± 0.17a | 0.3170±0.0001a |
12:12 | Day 19 | 83.04 ± 0.19b** (ab) | 0.2313±0.0001b | |
8:16 | Day 21 | 79.23 ± 0.21c**(ac) | 0.2010±0.0001c* (ac) |
Highest cell density and specific growth rate were recorded in all cultured species that were exposed to continued illumination (24:0. L/D) followed by 12:12 and 6:18 L/D respectively. Early stationary phases differed for
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1.07 | 0.35 | 0.30 | 0.34 | 0.17 | 0.25 | 0.41 |
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0.81 | 0.65 | 0.20 | 0.29 | 0.49 | ||
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0.65 | 0.96 | 0.23 | 0.19 | 0.51 | ||
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1.19 | 0.53 | 0.50 | 0.74 | |||
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0.20 | 0.87 | 0.77 | 0.61 |
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1.54 | 0.50 | 0.44 | 0.48 | 0.25 | 0.36 | 0.59 |
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1.16 | 0.94 | 0.28 | 0.42 | 0.70 | ||
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0.94 | 1.39 | 0.33 | 0.28 | 0.73 | ||
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1.72 | 0.77 | 0.72 | 1.07 | |||
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0.29 | 1.25 | 1.11 | 0.89 |
The 12:6:4 ratios showed the best average (74%) growth rates of natural increase at log phase. The second was 12:8:4 with 61% average growth rate.15:15:15 NPK ratio showed the lowest average growth rate of 41% (Table 4). The
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0.65 | 2.00 | 2.28 | 2.06 | 4.07 | 2.80 | 2.31 |
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0.86 | 1.06 | 3.55 | 2.38 | 1.96 | ||
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1.06 | 0.72 | 3.04 | 3.62 | 2.11 | ||
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0.58 | 1.31 | 1.39 | 1.09 | |||
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3.41 | 0.80 | 0.90 | 1.70 |
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15.56 | 47.99 | 54.84 | 49.55 | 97.66 | 67.32 | 55.49 |
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20.60 | 25.55 | 85.22 | 57.05 | 47.11 | ||
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25.44 | 17.28 | 73.01 | 86.79 | 50.63 | ||
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13.97 | 31.34 | 33.37 | 26.22 | |||
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81.76 | 19.14 | 21.54 | 40.82 |
Measurement of generation time for
Numerous nutrient media have been use for the culture of pure
Different diets gives significantly (P<0.05) different densities of
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0.1150 | 8 |
Baker’s yeast | 0.0756 | 11 |
The development times for nauplii, copepodite, adult and gravid female were observed separately using the copepod culture fed on
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Number of eggs | 3 | 21.33 ± 1.15 | 20 | 22 |
% hatching | 3 | 96.82 ± 2.77 | 95 | 100 |
Maturation time(days) | 3 | 1.33 ± 0.58 | 1 | 2 |
Generation time(days) | 3 | 20.67 ± 3.51 | 17 | 24 |
Production of the gravid females and the population density obviously increased when
A female of
4. Conclusion
Acknowledgments
The authors are grateful to the Ministry of Science, Technology and Innovation (MOSTI) for the funding given under the ABI-MOSTI grant (2009-2011), “Mass fry production technology for grouper (Epinephelus sp.)”, the Ministry of Education Malaysia for the Knowledge Transfer Programme grant (2012-2013) Using Microalgae and Copepod Live Feeds for Brackish Water Aquaculture Farm, UMT-PPKJBS.
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