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Cultivation of Algae in Vegetable and Fruit Canning Industrial Wastewater

Treatment Effluent for Tilapia (Oreochromis niloticus) Feed Supplement

Article  in  International Journal of Agriculture and Biology · April 2015

DOI: 10.17957/IJAB/17.3.14.502

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Tapana Cheunbarn Siraporn Cheunbarn

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
14–502/2015/17–3–653–657
DOI: 10.17957/IJAB/17.3.14.502
http://www.fspublishers.org

Full Length Article

Cultivation of Algae in Vegetable and Fruit Canning Industrial

Wastewater Treatment Effluent for Tilapia (Oreochromis niloticus) Feed
Supplement
Tapana Cheunbarn* and Siraporn Cheunbarn
Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
*
For correspondence: [email protected]

Abstract
This work was conducted to study the possibility of cultivating algae for use as fish feed using vegetable and fruit canning
industry wastewater treatment effluent. The results showed that Chlorella vulgaris showed successful growth in 10% initial
stock solution, 20 cm water depth and for 12 days of cultivation. The algae attained a cell density of 13.72 × 105 cells/mL,
biomass dry weight of 255 mg/L and protein content of 45.6%. Dried algae were used as a feed additive for tilapia
(Oreochromis niloticus L.) cultivated in a vegetable and fruit canning industry oxidation pond. After 3 months of cultivation,
fish fed 3% dried algae had a higher (P<0.05) fish survival rate and percentage weight gain than the fish fed only the
commercial feed. © 2015 Friends Science Publishers

Keywords: Chlorella vulgaris; Wastewater; Feed supplement; Tilapia

Introduction Materials and Methods

Vegetable and fruit processing can be considered an Wastewater Effluent Sample

important industry in agricultural countries, but considerable
amounts of wastewater are generated. Generally, wastewater The effluent was collected from a vegetable and fruit
is treated by anaerobic digestion followed by an aerobic (Lungan, Lychee, Rambutan, Bamboo shoot and baby corn)
wastewater treatment processes; however, after treatment, canning industrial effluent, which was treated by anaerobic
inorganic substances remain. This treatment effluent digestion followed by an aerated lagoon. The characteristics
contains a high amount of macronutrients such as of the effluent were: BOD 25‒35 mg/L, COD 65‒80 mg/L
nitrogen and phosphorus, which can cause and pH 6.5‒7.2. (APHA, 1998)
eutrophication when they come into contact with natural Algae Isolation and Purification
water resources. On the other hand, these substances can
be developed as important food sources for the Water samples were filtered and transferred to the
cultivation of algae with high economic significance. At laboratory. The streak plate technique was employed with
present, treated wastewater is used to cultivate many species Jaworski's medium under controlled conditions. The algae
of algae such as Spirulina sp., Chlorella sp. and cells were isolated under light microscope (Olympus, Model
Scenedesmus sp. (Gantar et al., 1991; Cheunbarn and CH30RF200) and identified as Chlorella vulgaris by
Peerapornpisal, 2010; Hongyang et al., 2011; Arbib et al., morphological such as colour, cell shape and size
2012; Wang et al., 2013). These algae can be developed into (Peerapornpisal, 2006; Peerapornpisal, 2013). The plate
animal feed, especially as a feed mixture supplement for culture was transferred to liquid medium to prepare a stock
fish to improve yield and quality (Promya and Chitmanat, solution.
2011; Ajiboye et al., 2012; Sing et al., 2014).
In this study, a microalgal Chlorella vulgaris isolated Suitable Condition for Cultivation of Algae
from vegetable and fruit canning industrial effluent was
investigated. The study focused on suitable conditions for The stock solution of Chlorella vulgaris (OD560 0.8) was
algal cultivation and use of the algae as a fish feed additive inoculated in 500 mL volumetric flask with 300 mL of the
for tilapia (Oreochromis niloticus Linn.). It could reduce the effluent. Four treatments with three replications of algal
capital cost of fish rearing, as well as serve as a method of culture with initial concentrations of 2, 5, 10 and 20% were
treating the effluent from this factory with the highest evaluated. The culture flasks were continuously aerated with
degree of efficiency. an electric aerator for 16 days. Cell growth was determined

To cite this paper: Cheunbarn, T. and S. Cheunbarn, 2015. Cultivation of algae in vegetable and fruit canning industrial wastewater treatment effluent for
tilapia (Oreochromis niloticus) feed supplement., Int. J. Agric. Biol., 17: 653‒657
 Cheunbarn et al. / Int. J. Agric. Biol., Vol. 17, No. 3, 2015

by cell counting with a hemocytometer, and the optical depending on procedure that has been described by
density was measured at 560 nm (Spectronic Genesys 5 APHA (1998). The four heavy metals, cadmium (Cd),
UV-visible spectrophotometer) every two days. copper (Cu), zinc (Zn) and mercury (Hg) were measured at
The suitable initial algal concentration was cultivated the end of the experiment by ICP-MS (Agilent, Model
in 100 L effluent in a 200 L cement pond for 15 days. The 7500A).
three different depths (20, 30 and 40 cm) with three
replications were evaluated. The cellular concentration was Statistical Analysis
determined every 3 days.
For mass culture, a suitable initial concentration of The differences of fish growth performances when use
algae, depth and retention time based on the previous study algae for fish feed supplement were analyzed. The study
was prepared in a 200 L cement pond with a 100 L working was divided into two treatments with three replications.
volume. The algal biomass was harvested, dried and The comparison and derivation were made between
analysed for the nutrient value according to the AOAC treatment 1 (0% algae) and treatment 2 (3% algae).
method (AOAC, 1984). Independent t-tests by compare means was performed using
SPSS 16.0 for windows. P<0.05 was considered statistically
Feed Supplement for Tilapia significant.

Dried algae were ground into a powder, mixed with Results

commercial fish pellets and coated with fish oil. The
samples were kept in a plastic bag, after drying in the open Suitable Condition for Cultivation of Algae
air. Tilapia (Oreochromis niloticus Linn.) were cultivated in
cages (3.0×3.0 × 1.5 M) for 3 months in a vegetable and In this study, it was shown that algal growth was maintained
fruit canning oxidation pond. The study was divided into in a stable manner, as evidenced by the increasing OD value
two treatments with three replications. Treatment 1 and number of cells. The highest growth was found at 10%
consisted of commercial fish pellets (CP brand product) initial algae concentration. A highest cell density (16.08 ×
mixed with 0% dried algae, while treatment 2 was 105 cells/mL) was found on day 16 of the experimental
commercial fish pellets mixed with 3% dried algae. The fish period with an OD value of 0.42. This was followed by 2, 5
were fed twice a day (09:00‒10:00 am and 3:00‒4:00 pm). and 20% initial stock solution, which produced highest cell
The quantity of feed was adjusted based on the monthly density of 13.83 × 105 cells/mL, 13.39 × 105 cells/mL and
weight of the fish. The monthly weight was recorded and 12.43 × 105 cells/mL, respectively (Fig. 1 and 2). The
the feed supplied was used to compute the growth and suitable depth for algal growth was 20 cm. A highest
nutrient utilisation parameters as follows: number of cells was observed on day 15 at 18.14 × 105
cells/mL with an OD value of 0.546. This was followed by
1. Specific growth rate (%/day) = (ln W2– ln W1/no. of 30 and 40 cm depth, which produced highest cell density of
days during experiment) ×100
12.42 × 105 and 6.23 × 105 cells/mL, respectively (Fig. 3
Where W2 = final weight of fish, W1= initial weight of and 4). The best time point for harvesting was on day 12
fish. when the algae were completely mature.
The mass culture was done in batch system by the
2. Survival (%) = F1/F2×100 suitable condition with 10% initial stock solution, 20 cm
Where F1 = number of fish at the beginning of the depth and harvesting was done after 12 days, i.e. during the
experiment, log phase. After cultivation, the algae had an OD value of
F2 = number of fish at the end of the experiment. 0.428, cell density of 13.72 × 105 cells/mL, and dry weight
of 255 mg/L. The nutritional properties were protein 45.6%,
3. Feed conversion ratio (unit) = feed intake (g)/fish fibre 8.5%, ash 17.0%, fat 15.2% and carbohydrate 10.6%
weight gain (g) of dry weight.
4. Percentage weight gain (%) = (W2 – W1)/W1×100
Where W1 = initial weight of fish, W2= final weight of fish. Fish Growth Performance on the Algae Feed
Supplement
Water Quality during Fish Cultivation
Feeding tilapia with commercial pellets and commercial
Water quality was measured every two weeks during the pellets mixed with 3% dried algae, led to a significant
cultivation period. Temperature and pH were measured by difference in the fish survival rate and percentage weight
multi parameter (Eutech instrument, Model PCTestr35). gain at the 95% confidence level (P<0.05). There were no
Nitrate, ammonia, and phosphate were measured by UV- significant differences between Treatments 1 (0% algae)
Visible spectrophotometer (Hach Odyssey, Model and 2 (3% algae) in the specific growth rate and feed
DR/2500). DO, BOD, COD and TS were measured conversion rate (Table 1).

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Table 1: Growth performance of tilapia in an experimental pond using Chlorella vulgaris as feed supplement for three
months

Parameters Treatment 1 (commercial fish pellet+0% algae) Treatment 2 (commercial fish pellet + 3% algae) P-value
Specific growth rate (%/day) 2.16±0.006 2.37±0.152 ns
Survival rate (%) 61.7±9.5 76.3±5.5 *
Feed conversion rate (FCR ) 0.45 ±0.05 0.40 ±0.03 ns
Percentage weight gain (%) 597.6±55.19 740±91.32 *
Mean± standard deviation. *= significant statistical differences (P<0.05), ns= no significant statistical differences

Table 2: Water quality during tilapia cultivation

Water Quality Index Week 0 Week 2 Week 4 Week 6 Week 8 Week 10 Week 12 mean ±SD
Temp (ºC) 23.6 23.4 24.5 25.6 24.2 24.6 24.4 24.3±0.7
TS (mg/L) 67 64 79 76 56 65 64 67.3±7.8
pH 7.5 7.2 6.9 7.6 7.5 6.9 7.4 7.3±0.29
DO (mg/L) 5.5 5.2 5.5 6.5 6.5 5.8 5.4 5.8±0.5
COD (mg/L) 68 80 76 65 82 75 79 75.0±6.3
BOD (mg/L) 16 18 19 15 14 19 20 17.3±2.3
NO3-- N (mg/L) 17 19 14 25 16 14 19 17.7±3.8
NH3-N (mg/L) 0.14 0.16 0.23 0.15 0.30 0.24 0.23 0.2±0.06
PO4 3- (mg/L) 0.11 0.32 0.16 0.22 0.30 0.23 0.18 0.22±0.07

Discussion
A suitable initial concentration is considered necessary for
cultivating algae. A huge number of algae are unable to
survive under inappropriate conditions although they are
native ones and adapted to local environment. This research
found that the suitable Chlorella vulgaris initial stock
concentration was found at 10% (OD560 0.8). If the initial
concentration is too low, only a few algae would continue to
grow because of breaking colonies caused by photo-
oxidation (Abeliovich and Shilo, 1972; Benchokroun et al.,
2003; Baroli et al., 2004). This situation occurred at the 2
Fig. 1: Growth (cell densities) of Chlorella vulgaris and 5% initially stock solution.
cultivated at various initial concentrations Also, if the initial concentration is too high, the yield
would not be good because they would obstruct light from
each other, and photosynthesis would be reduced (Gitelson
et al., 1996). This situation appeared at the 20% initially
stock solution, showing rapid growth only at the beginning
but after 10 days of the experiment the growth slowly
declined. Sedimentation also occurred, as the green colour
of algal biomass was observed at the bottom of the
container.
It was found that algae cultivated at 20 cm depth
showed the best growth rate. During day 0‒3, the algal
growth was slow and increased only slightly because the
algae were adapting to the new environment and had not yet
started to increase in numbers. After the initial adaptation,
Fig. 2: Growth (OD) of Chlorella vulgaris cultivated at the algae started to grow quickly during days 3‒12, then
various initial concentrations grew slowly again until they reach a stationary phase (Fig. 3
and 4). However, the best time point for harvesting was on
Water Quality During Fish Cultivation day 12 when the algae were completely mature, only a few
dead cells were found together with a lower level of
The water quality results taken during the 3 months of
chlorellin. Chlorella vulgaris cultures usually release
rearing tilapia in the oxidation pond are shown in Table 2.
chlorellin (Pratt et al., 1945), a substance similar to a fatty
Four heavy metals were measured. The results showed that
only zinc was measured at a very low level of 0.01 mg/L, acid and a hydrocarbon (Spoehr and Milner, 1949) having
and the others were not found. allelopathic activity, a property that allows it to inhibit the

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 Cheunbarn et al. / Int. J. Agric. Biol., Vol. 17, No. 3, 2015

the dry cell weight of the algae was 0.541 g/L after
cultivation in BBM medium, the dry weight was 0.629 g/L
(Toyub et al., 2007), and in the Trebon system, the production
rate for Chlorella was 10 g/L (Doucha et al., 2005).
Normally, when algae are cultivated in wastewater,
they are widely accepted as a feed supplement, especially for
fish feed. However, the present study found that FCR in the
both treatments were very low (Table 1), indicating that they
took less feed to produce 1 kg of fish. Both fish diet formulas
were not the only kind of feed for the fish, because the fish
were in the oxidation pond where inorganic substrates were
still high, so plankton could grow. The plankton became a
natural food for the fish, so it was possible to use less feed to
produce 1 kg of fish. However, the amount of algae as a
Fig. 3: Growth (cell densities) of Chlorella vulgaris protein supplement for fish depends on the type of fish. For
cultivated at various depths example, Paralichthys olivaceus grew best when fed with
2% Chlorella powder (Koo et al., 2001), while Sebastes
schlegeli (Hilgendorf) grew best when fed with 0.5%
Chlorella powder (Bai et al., 2001).
Normally, when algae are widely accepted as a feed
supplement, the supplementation would also increase the
organic substances in the wastewater affecting the quality of
the effluent. However, the analysis of water quality showed
that the amounts of heavy metals were below the standard
for inland aquaculture (MNRE, 2007) and the standard for
industrial water effluents (MOSTE, 1996).

Conclusion

In conclusion, the agro-industrial effluent could be applies

Fig. 4: Growth (OD) of Chlorella vulgaris cultivated at for microalgae cultivation, especially the strain that isolated
various from the effluent. With high protein and many nutrients
contents, the microalgae could be applies as a feed
growth of other living organisms such as bacteria and algae supplement for fish to improve yield quality.
(McCracken et al., 1980). Moreover, it has been found that
chlorellin at lower concentration still allows algae to grow. Acknowledgement
However, if chlorellin is present at a concentration greater
than 6.5 mg/L, it affects growth (DellaGreca et al., 2010). The authors would like to acknowledge National Research
Chlorellin extracted from aged Chlorella sp. is still able to Council of Thailand for supporting this work.
obstruct photosynthetic activity (Mandalam and Palsson,
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