MATERIALS AND METHODS

Production of PNSB biomass: The strain of phototrophic bacterium Rhodovulum sp. was isolated locally from the seabass broodstock tank at the hatchery of Borneo Marine Research Institute, Universiti Malaysia Sabah. The synthetic 112 media was used for the inoculum preparation as well as for the mass production of PNSB biomass (Ronald, 1996). A 5% inoculum (v/v) of Rhodovulum sp. was inoculated into a 2-L bottle contained sterilized 112 media. The bottles were incubated under 2500 lux light intensity at a temperature of 30±2°C under anaerobic condition for 48 h. The bacterial cultures were centrifuged to obtain biomass. The harvested fresh biomass of Rhodovulum sp. was used in the diet preparation.

Fish feed preparation: The commercial seabass sinking feed (Cargill Feed Sdn. Bhd., Malaysia) was used in this experiment. The feed were ground into powder using a grinder and PNSB biomass was included at 0.3, 0.6 and 0.9% of the diets. A commercial feed binder, carboxymethyl cellulose (1%) was also added in this mixture to bind all ingredients. Water was then pour into the mixture and mixed well to form dough. The dough was screw-pressed through a 3 m die and the strands of feeds were air-dried overnight. The control diet was without the addition of PNSB biomass. The experimental diets were refrigerated until used.

Feeding trial: A total of 12 cylindrical cages (61 cm depth and 43 cm diameter) were randomly placed in a 15-tons circular polyethylene tank with flow-through system and continuously aerated. Temperature, salinity, pH and dissolved oxygen were measured every day and maintained at 27.9±1.1°C, 32.6±1.8 ppt and 7.4±1.6 and 6.5±0.4 mg L^-1, respectively. Fish were acclimatized to the experimental cages for two weeks and fed a commercial seabass diet before the start of the experiment.

The triplicate fish groups (15 individual per cage) of initial body weight 6.5±0.5 g were fed experimental feed at apparent satiation level by hand twice daily. The total amount of given feed was recorded every day. Fish was individually weighed and total length was recorded at the beginning and end of feeding trial. Bulk weight of the fish was recorded once a week. The feeding trial was conducted for 12 weeks.

Proximate composition analysis: Experimental feeds and whole-body fish were subjected to proximate analysis using standard methods. Experimental feed and fish carcasses were blended, oven-dried and ground into powder before proximate analysis. Moisture, crude protein, crude lipid, crude fiber and crude ash were determined using methods described by AOAC (1990).

Calculation and statistical analysis: Growth performance, Feed Conversion Ratio (FCR), survival and condition factor were calculated as follows:

One-way ANOVA was used to compare growth performance, FCR, survival, condition factor and whole-body proximate composition. Homogeneity of variances was tested with Levene’s test and multiple comparisons among treatments were performed with a Tukey HSD post hoc test. Significance level was set at p<0.05. Statistical package SPSS v.11.0 for Windows was used for all statistical analyses.

RESULTS AND DISCUSSION

Analysed proximate composition of experimental feeds is presented in Table 1. There was no significant different (p>0.05) detected in the proximate composition of the experimental feeds. Moisture, crude ash, crude lipid and crude protein ranged from 12.34-12.47%, 11.13-12.03%, 7.50-8.12% and 38.12-39.18%, respectively. These values correspond to the proximate composition provided by the feed manufacturer (moisture 11%, crude ash 11%, crude lipid 8.0% and crude protein 45%). At the end of feeding trial, fish fed diet with 0.3% PNSB biomass obtained the highest weight gain (419%), followed by the control feed (373%), feeds with 0.9% (363.9%) and 0.6% (363.3%) Rhodovulum sp. biomass. Specific growth rates were also following the weight gain trend with values ranged from 1.46 (0.6% PNSB) to 1.87 (0.3% PNSB). The best FCR was also obtained in feed with 0.3% PNSB (1.95) (Table 2). The FCRs in other feeds ranged from 2.04-2.15. In term of survival rate, fish fed control feed resulted in the lowest survival rate with 73.3%. Survival rates in other treatments were 80-86.7%. Similarly, condition factor was not affected by the dietary treatments (Fig. 1).

Table 1:	Analysed proximate composition of experimental feed on % dry matter basis

Table 2:	Growth performance and feed conversion ratio of experimental fish at the end of feeding trial
SGR: Specific growth rate, FCR: Feed conversion ratio

Table 3:	Whole-body proximate composition of experimental fish at the end of feeding trial on % dry matter basis

Fig. 1:	Survival rate (%) of fish fed experimental feed after 12 week feeding trial

Whole-body proximate composition of fish is shown in Table 3. Inclusion of PNSB in the feed of seabass juveniles did not significantly (p>0.05) affect the body proximate composition.

In general, the growth of juvenile seabass in the present study is similar to the growth obtained in our separate study on the use of alternative oil in seabass feeds (Shapawi et al., 2011). Even though there were no significant differences detected in the growth performance, FCR and survival rate, the fish fed 0.3% PNSB consistently giving better values in all mentioned parameters. It is interesting to note that higher PNSB inclusion (0.6 and 0.9%) has resulted in poorer fish performance than in control feed and 0.3% PNSB. This finding suggested that high level of PNSB biomass in fish feed might not give much benefit to fish growth. This is in agreement with a finding by Azad et al. (2002) which reported a growth benefit of phototrophic bacteria in juvenile tiger prawn (Penaeus monodon) when included at only 0.1% of the feed. However, in clusion of probiotic in the feeds might have some influence in the survival of the target fish. In the present study, the survival rate of fish fed the control feed was about 10% lower than the fish fed feed with PNSB. This can be considered a significant contribution in lowering the cost of feed production since only little amount of PNSB biomass is required to promote the growth performance and survival of cultured fish.

Little is known about the potential of PNSB as a formulated feed supplement in cultured fish. Several studies reported the positive effects of other species of probiotics on growth performance of cultured aquatic animals when included in the feeds such as in Asian seabass (Rengpipat et al., 2008), tilapia (El-Haroun et al., 2006; Chantharasophon et al., 2011), African catfish (Abdelhamid et al., 2009) rabbit fish (El-Dakar et al., 2007), rainbow trout (Merrifield et al., 2010a, b), giant freshwater prawn (Deeseenthum et al., 2007) and penaeid shrimp (Fernandez et al., 2011). Merrifield et al. (2010b) was also reported a similar finding to the present study in term of whole-body proximate composition, where no significant different was observed as a result of probiotic inclusion in the feeds. In contrast, Bagheri et al. (2008) reported that rainbow trout fed feed supplemented with probiotics resulted in higher protein and reduced lipid levels in the carcass.

In general, the findings from the present study suggested that Rhodovulum sp. might be able to promote growth, FCR and survival of cultured fish when included at low level of below 0.4%. Further research on the lower inclusion level (<0.3% of the diets) of PNSB deserved an investigation.

ACKNOWLEDGMENTS

We would like to thank the staff of Fish Hatchery of Borneo Marine Research Institute, Universiti Malaysia Sabah for their assistance in the feeding trial.