Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 3 (march 2020) : 310-316

Comparison of fertilization prototype on biofloc development and its characteristics in GIFT Tilapia Culture

M. Menaga2, S. Felix1,*, M. Charulatha2, C. Mohanasundari2, A. Gopalakannan2
1Tamil Nadu Dr.J.Jayalalithaa Fisheries University, Nagapattinam- 611 002, Tamil Nadu, India.
2Department of Aquaculture, Dr. M.G.R. Fisheries College and Research Institute, Ponneri-601 204, Tamil Nadu, India.
Cite article:- Menaga M., Felix S., Charulatha M., Mohanasundari C., Gopalakannan A. (2019). Comparison of fertilization prototype on biofloc development and its characteristics in GIFT Tilapia Culture . Indian Journal of Animal Research. 54(3): 310-316. doi: 10.18805/ijar.B-3776.

ABSTRACT

A study was conducted to evaluate the effect of different fertilizers in GIFT Tilapia culture using biofloc technology. Animals (5±0.23g) were stocked at a density of 30m-3 in 500 litres FRP tanks and spentwash was used as a carbon source to maintain a C:N ratio of 10:1 for 42 days. The experimental group includes fertilization using ammonia sulphate alone (T1) and fertilization using different inorganic fertilizers (T2). No significant differences in FCR, specific growth rate, weight gain and survival of animals were found between the treatments. Proximate composition and fatty acid profile of floc were comparatively rich in T2. Increased solid concentrations with higher Floc volume index and floc sizes were recorded in T2. The rapid floc development along with multiplication of heterotrophic bacteria and decreased vibrio population was observed in T2. The present study confirmed the influence of fertilizers on the physical and nutritional quality of biofloc in GIFT tilapia culture.

INTRODUCTION

The intensification of the aquaculture can be driven by many advanced technologies and one among them was the biofloc technology. This technology allow aquaculture animals to grow at higher stocking densities with minimal water exchange. The nitrogen recovery in the biofloc technology aids in the prevention of disease outbreak by providing proper biosecurity. Unlike Recirculatory aquaculture systems this technology does not require any external filtration rather dense microbial biomass, strips the ammonia inturn to serve as a nutritional source. Tilapia is a hardy species and its omnivorous feeding habit accommodates this fish in the intensive culture practices. Its tolerance to the wide environmental conditions has exceeded the production of 5 million tonnes per year with steady growth rate of 5-8 percent globally (Menaga and Fitzsimmons, 2017).The suspended growth in ponds such as phytoplankton, bacteria, live and dead particulates and grazers constitutes the floc. Their growth is influenced by various physico chemical factors such as Temperature, pH, Salinity and aeration of the culture systems. The growth and stability of the flocs formed can also be determined by using different carbon sources and at various C/N ratios. The growth of heterotrophic bacteria can be promoted by the external supplementation of carbon sources such as dextrose, sugar, rice flour, wheat flour, rice bran, molasses etc. However the use of distillery spentwash as a carbon source in the biofloc technology is limited. The synergistic way of utilization of distillery spent wash (DSW) by the microbes for its effective degradation brings an eco-friendly application in aquaculture. The ability of the DSW in rapid release of the carbon content paves the way for the growth of heterotrophic bacteria to assimilate ammonia with decreased concentrations of TSS. Assimilation of ammonium by heterotrophic bacteria takes place rapidly due to the faster growth rate as heterotrophic biomass yield per unit substrate are a factor of 10 higher than autotrophic bacteria (Hargreaves,2006).The use of distillery spentwash as a carbon source has been recommended for shrimp as well as GIFT Tilapia (Menaga et al., 2017; Yuvarajan et al., 2018). The balance of carbon and nitrogen with the external supplementation of carbon will convert the ammonium and other organic nitrogenous waste into bacterial biomass (Schneider et al., 2005). The chemo-autotrophic system exhibits the classic increase and sudden fall out in ammonia concentration as the nitrifying bacteria oxidize the nitrite-nitrogen to nitrate-nitrogen with the help of available CO2 in the systems. Another disadvantage of nitrifying bacteria includes its higher sensitivity to the increased ammonia and decreased dissolved oxygen level which in turn impacts the maintenance of optimum water quality parameters (Masser et al., 1999; Villaverde et al., 2000; Ling and Chen, 2005). In addition, both the heterotrophic and the chemoautotrophic show very low concentrations of nitrite-nitrogen, because nitrite is not a product in either pathway.The biofloc technology has been implemented in various countries and its adoption strategies vary for different countries. The fertilization prototype adopted by the farmers on biofloc has not been discussed much so far. The use of inorganic fertilizers such as urea, triple super phosphate has been in practice as a source of nitrogen and phosphorus in the aquaculture systems. As the pond fertilization augment the production of plankton thereby the different trophic levels including autotrophic and heterotrophic bacterial population helps in increased fish production (Grag and Bhatnagar, 2000). The objective of the present study was to assess the different methods of fertilization prototype for the biofloc development using distillery spentwash as a carbon source at constant C: N ratio 10:1 in the tank culture of GIFT Tilapia.

MATERIALS AND METHODS

The experiment consists of two treatments such as fertilization of water using 20 gm L-1 pond soil, 10 mg L-1 ammonium sulphate and 200 mg L-1 distillery spentwash as described by (Avnimelech and Kochba, 2009) (T1) and fertilization of water using various inorganic fertilizers as suggested by (Taw, 2006) (T2) in freshwater (0 ppt). The list of fertilizers used in T2 study were given in the Table 1. Distillery spent wash (DSW) was used as the carbon source to maintain the C:N ratio at 10:1 and it was added thrice to the culture tanks following standard protocols of Crab et al., (2010) with slight modification based on the carbon content of Distillery spentwash. The DSW used in the current study was collected from M/s.Rajshree Biosolutions Private Limited, Coimbatore and stored in room temperature. The main characteristics of DSW were analysed at Central Leather Research Institute, Chennai and the results were listed in Table 2. The GIFT Tilapia fingerlings (5±0.23g)  were stocked at a density of 30/m3 in 500m3 FRP tanks in triplicates .Animals were fed with 30% crude protein feed daily as per their body weight for 42 days. The water quality and floc characteristics were investigated throughout the culture trial.
 

Table 1: Fertilizers used for biofloc development.


 

Table 2: Physico-chemical properties of Distillery Spentwash.


 
Growth parameters
 
The growth parameters of GIFT Tilapia were monitored on weekly basis and various growth indices were calculated:
 
Feed conversion ratio = Feed given /Body weight
Specific growth rate (%) = Ln (final weight) –Ln (Initial weight) x 100 /Number of days
Survival rate (%) = Total number of Fish harvested/Total number of Fish stocked x 100
 
Water quality parameters
 
Temperature (YSI, ProDSS Multiparameter) and pH (Labtronics instruments) were measured daily. Dissolved oxygen, free carbon dioxide, salinity, alkalinity, hardness, calcium and magnesium ion concentration were measured weekly as per APHA (2008). Water samples were filtered using No.1 Whatman filter paper and collected filtrate was analysed using Resorcinol method for nitrate-N (NO3 -N)  and nitrite (NO2 -N). Phenol hypochlorite method was used for total ammonia nitrogen (TAN) and Orthophosphate (APHA, 2008) were recorded weekly once. Total heterotrophic bacteria (THB) and Vibrio were estimated and expressed as colony forming units (CFU) on weekly basis as per Bergey’s manual of systematic bacteriology (Holt et al., 1989).
 
Floc parameters
 
Biofloc water was collected using Imhoff Cone and it is kept undisturbed for 20 minutes for the floc to settle down. After 20 minutes the particles settled at the bottom was measured as floc volume (Avnimelech and Kochba, 2009). Floc porosity was calculated by the volume of water and floc settled in Imhoff Cone according to Smith and Coakley, (1984).
 
                                Porosity = (FV/WV)× 100
 
       
Floc volume index was obtained using floc volume and floc concentration (TSS) according to Mohlman (1934). It is calculated using the formula
 
             FVI = Floc volume (ml)/ Floc concentration (g)
 
       
Floc density index was calculated using floc volume index and it is the grams of floc which occupies a volume of 100 ml after 30 minutes of settling (WHO international reference centre, 1978).
 
                                                        FDI = 100/FVI
 
       
Total organic carbon was analysed according to Walkley and Black (1934).  From the titre value TOC was calculated according to the formula,
 
                TOC % = [10- (10 × y/x)] × 0.003 × 100/Vs
Where,
 x   = Titrite value of blank;    
y = Titrite value of sample;
Vs = Volume of sample
       
Floc size and shape were recorded under digital microscope (Lawrence and Mayo, NLCD-120e). Floc settling velocity was determined using Megara et al., (1976). BOD,TS,TDS,TSS and VSS  were determined according to APHA (2008) and expressed in mg L-1.These quantitative and qualitative characteristics of floc were determined weekly once during the culture trial.
 
Biochemical composition of biofloc
 
Proximate composition of the floc were performed as per standard method of AOAC (2005). Extraction of lipid from the biofloc was done as per Folch et al., (1957) with slight modifications and fatty acid analysis of the floc was analyzed in GC-MS at Veterinary College and Research Institute, Nammakkal.
 
Statistical analysis
 
Water and Floc quality were analysed using one way ANOVA between treatments at 5% level of significance. Growth parameters of the culture animals were analysed using one way ANOVA and post hoc analysis using Duncan Multiple range test for the significant values. Statistical analysis were performed using SPSS software version 20.0.

RESULTS AND DISSCUSSION

There was no significant difference in Temperature, Salinity, Dissolved oxygen, pH, Hardness, Magnesium, Ammonia-N, Nitrate-N and Phosphorus. The nitrite concentration pattern was different for both the treatments. Accumulation of nitrite concentrations was seen at the end of the second week in T1 and this may be due to the slow growth of heterotrophs in T1 than T2. The low concentrations of nitrite in T2 reveals the complete assimilation of ammonia to nitrate by heterotrophic bacteria under the similar environmental conditions (Ebeling and Timmons, 2007). In the present study, optimum NH3-N and NO3-N concentrations were observed in all the treatments as cited by Hargreaves, 2013 and their accumulated concentrations did not vary with the effect of carbon supplementation. The concentration of CO2 was relatively higher in T2 (4-4.9 ppm) and this may be due to the ammonia regeneration by the bacterial biomass under biofloc culture systems (Avnimelech, 2015). Total alkalinity and calcium concentration found significantly different between the treatments. The lower level occurrence in T2 may be due to the buffering action of bacteria as reported by Ebeling et al., (2006).
       
In both treatments the pH and temperature were within the desirable ranges favouring the growth of the culture species (Table 3) (Crab, 2009). The presence of vibrio bacteria population was seen in both the treatments, however inclined multiplication rate was found in T1 compared to T2. This may be due to the faster replication rate of heterotrophs compared to slower growth rate of autotrophs (Ebeling et al., 2006).
 

Table 3: Water quality parameters of experimental groups for the 42 days of culture trial.


       
The mean final weight, survival and FCR values for the fertilization study were presented in Table 5. Based on the statistical analysis; there was no significant differences between the treatments however improved final mean weights of the fishes in T2  culture tanks was recorded. This may be due to the enhanced floc production and thereby the consumption of floc lead to increased weight gain.
 

Table 5: Growth Parameters of GIFT Tilapia of the experimental groups at the end of the culture trial.


               
The small changes in the floc will be highly influenced by the ionic composition of the culture water and its relative changes in the ionic strength will bring a substantial change in the floc morphology. The other factors influencing the physical and chemical characteristics of biofloc includes the type of carbon source, C/N ratio, DO concentrations, shear force caused due to aeration and settling time. The fluctuations of floc quality characteristics throughout the culture trial was given in Fig 1 and Table 6.
 

Fig 1: Quantitative and Qualitative Characteristics of biofloc in the experimental groups of GIFT Tilapia culture.


 

Table 6: Quantitative and Qualitative Characteristics of biofloc in the experimental groups of GIFT Tilapia culture.


       
The concentration of Total solids, Total suspended solids and Total dissolved solids found to be higher in T2 than T1.This may be due to the addition of grain pellets and carbon source in the prototype of T2 which paves the way for the faster development of biofloc.Also the percentage of nitrogen in urea constitutes about 45% and in ammonium sulphate it is closely to 20% (Boyd, 2012). This leads to the presence of increased floc volume in T2 than T1.The inclined trend of BOD level in T2 can be correlated with the abundance of the heterotrophic microorganisms. Significant difference was found between the treatments in total plate count and vibrio count of the culture water (Table 4). The increase in total plate count in T2 with decreased vibrio count revealed the multiplication of beneficial heterotrophic organisms in the culture water. The increased oxygen demand of microbes, culture animal and the organic matter was vividly observed in T2 with the constant C/N ratio under vigorous aeration.
       

Table 4: Total Heterotrophic bacteria and Vibrio count in culture water of the experimental groups.


 
Due to the increased solid concentrations in T2 the presence of higher organic particulate particles was observed with higher Volatile suspended solids. The total organic carbon content of the floc is directly proportional to the VSS and hence the amount of total organic carbon was found higher in T2. The floc size of 250-1200 µm was recommended as it serve as a nutrition source for the aquaculture animals (Barros and Valenti, 2003). The floc sizes observed in the study were within the desirable range for the intake of GIFT Tilapia and a steady state was observed in the treatments based on the floc volume. The floc porosity was higher in T1 compared to T2. The floc porosity is indirectly proportional to the floc size as the flocs are highly porous, the filtration of the suspension will be higher and hence porosity of smaller sized flocs will be higher than larger flocs. The floc volume index (FVI) was found to determine the settleability as well as the age of the floc. Lower the FVI higher the settleablity and it can improve the performance of the culture systems as a source of nutrition thereby maintaining the optimum water quality parameters.
       
The higher FVI was observed in T2 and this pave more opportunity for the animal to filter the floc with improved floc intake. However the higher FVI also would cause possible clogging of fish gills was also observed as suggested by Avnimelech (2012). The better settleability of floc was observed in T1 due to decreased floc volume and lower FVI. The floc density index (FDI) is an index to determine the compaction and settling ability of floc. The flocs in T1 possessed higher FDI with good compact ability compared to T2. However these changes were observed in a very small level.
       
In the proximate composition, crude protein, crude lipid and total ash were significantly different (p<0.05) between the treatments. This may be due to the rapid formation of floc leading to increased floc volume in T2 by the use of different inorganic fertilizers. These results also indicate the fact that the nutritional quality of floc was not only related to  protein content of the feed and carbon source but also on the use of fertilizers triggering the growth of favourable micro-organisms in the  culture systems.
       
The proximate analysis of the biofloc obtained from both treatments were similar to the nutritional profile of floc developed using wheat flour and molasses as carbon sources (Ballester et al., 2010). The crude protein content of biofloc collected from two treatments were within the range and confirmed the use of biofloc as a nutritional source to the culture animals (Azim and Little, 2008; De Schryver and Verstraete, 2009; Crab et al., 2010; Ekasari et al., 2010).
       
The ash content in the floc may vary based on the accumulation of solid concentrations in the biofloc systems. As the suspended and dissolved solid concentrations were higher in T2 the increased ash content (45%) was found in T2 which is similar to the findings of De Schryver and Verstraete (2009). It is interesting to note the increased gross energy level was observed in the treatments and this may be due to the use of Distillery spentwash as carbon source. The dominant fatty acids such as palmitic acid, linoleic acid and oleic acid in the biofloc samples of T1 and T2 were recorded. The optimum level of Omega 3 fatty acids were observed in T1 and T2 and lower level of PUFA was found in T1 compared to T2.The difference in PUFA level may be due to the huge heterotrophic bacterial population which acts as a nutrient source for the animals to consume (Meyers and Latscha, 1997). The multifold increase of heterotrophic bacteria improved the growth of GIFT Tilapia juveniles in T2 than T1 (Table 7). By the use of different inorganic fertilizers along with dolomite supplementation positively promoted the floc concentration for the maintenance of desirable water quality parameters. In addition the use of grain pellets also triggers the proliferation rate of bacteria for the optimum utilization of nutrients in the biofloc systems. The results of the present study confirmed the use of different inorganic fertilizers as an effective method for the biofloc production in GIFT Tilapia culture tanks.
 

Table 7: Nutritional Composition of biofloc obtained from the experimental groups in the GIFT Tilapia culture.

CONCLUSION

The biofloc developed from the different fertilization prototypes influenced the physical and nutritional quality of the floc by favouring the utilization of major nutrients from the fertilizers in the culture tanks. The stability of the flocs and its characteristics vary for the initial days of the culture for the two methods of fertilization however, the steady state floc quality maintenance after thirty days of the culture remained the same. The development of floc was very rapid and increased floc volume in the use of combination of different inorganic fertilizers throughout the experiment was observed. The lower floc volume was maintained in the ammonium sulphate based fertilization in the freshwater. Thus the findings of the study suggested the adoption of use of inorganic fertilizers for the faster development of floc with the predominant heterotrophic bacterial community. The floc stability and floc volume maintenance can be monitored with the addition of the distillery spentwash for the optimum water quality to improve the animal performance. The present findings can be further studied in different salinities as this biofloc technology is applied in both fresh water and sea water.

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