International Journal of Aquaculture, 2016, Vol.6, No.10, 1
-
9
6
these type bacteria can lead to the development of low - cost formulated diets such as the use of plant - based diet,
as 80% of total production cost is coming from feed.
In conclusion, a total of 7 bacteria associated with gastrointestinal tract of hybrid abalone displayed a capacity to
degrade cellulose. Among 7 endosymbiont bacteria, 2 isolates with the highest degrading activity were identified
as
Stenotrophomonas
sp and
Bacillus
sp
.
Further
in vitro
characterizations indicated these bacteria showed to be a
potential probiotic candidate for aquaculture species: had high viability in seawater, high survival rate in low pH
of a stomach simulation, resistant to the bile salt content in simulated intestinal condition, as well as non - toxic to
the cultured animal. These results may suggest that these bacteria are potential for aquaculture probionts; therefore,
effect of these bacterial supplementation on the food digestibility of aquatic species needs to be further studied.
3 Materials and Methods
3.1 Isolation of cellulose - producing bacteria
Cellulose - degrading bacteria were isolated using an enrichment technique according to a protocol developed by
Gupta et al. (2012) with slight modification. Briefly, gastrointestinal tract (GIT) of hybrid abalone was pooled and
homogenized with a stomacher. Thereafter, the homogenate was inoculated to a broth medium containing; 0.5 g.
L
-1
KH
2
PO
4
, 0.25 g. L
-1
MgSO
4
, 2 g. L
-1
carboxyethyl cellulose (CMC), and 2 g. L
-1
gelatin. All materials were
dissolved in distilled water and pH was adjusted to 6.8–7.2. After 7-day incubation, 1 mL aliquot was serial
diluted and 0.1 mL of each dilution was plated on the same medium with the addition of 15 g. L
-1
agar, followed
by 2-day incubation aerobically at room temperature. Colonies with apparently different morphological
appearance were transferred by streaking repeatedly on the agar plate for purification. The pure colonies were
preserved in 15% glycerol stock and stored in -80
o
C. The presence of cellulolytic activity was detected by
subculturing the pure bacterial isolates onto the CMC agar plates with 0.2 g. L
-1
Congo - red as colour indicator.
After 2-day incubation, colonies which showed discoloration of Congo-Red were taken as positive
cellulose-degrading bacteria, and only these isolates were further studied.
3.2 Quantification of cellulolytic activity
Quantification of cellulolytic activity was performed according to a protocol developed by Gupta et al. (2012)
with slight modification. Cellulose - degrading isolates were subcultured in 3 g. L
-1
CMC broth and incubated at
room temperature (21 ±0.5
o
C) for 7 days. Cells - free supernatant was harvested by centrifugation at 5 000 rpm
for 15 min at 4
o
C. Then, one mL of the cultured supernatant was mixed with 1 mL of 3, 5-dinitrocalicylic acid
solution (DNS) (0.2% phenol, 1% sodium hydroxide and sodium sulfite) and boiled for 5 min. After cooling at
room temperature, the absorbance of the mixed solution was measured with a Tecra infinite 200Pro
spectrophotometry at 540 nm wavelength. As a control, dilutions with several ranges of glucose concentration
were prepared. The results were expressed as mg glucose. L
-1
. Two bacterial isolates with the highest cellulolytic
activity was further studied on their adaptability in feed pellet, rearing water and simulated intestinal tracts.
3.3 Bacterial identification
The pure bacterial isolates with cellulolytic activity were subjected to standard phenotypical assays including
gram - staining, catalase, and oxidase production. In addition, the bacterial identity was confirmed using a colony
polymerase chain reaction (PCR) as described by Amin et al. (2016). Purified PCR products were sent for
sequencing, and the sequenced isolates were compared to published sequences using the BLAST search algorithm
to identify the isolates. In addition, the 16S rRNA gene sequence of these bacteria and several 16S rDNA
sequences of the closest-known strains derived from GeneBank were aligned with Genious software version 5.3.6
to show relative position of bacterial isolates using a Joining – neighboring method.
3.4 Survival in seawater
Fresh colonies of cellulose - degrading bacteria were picked and suspended in sterile phosphate buffer saline (PBS,
pH 7.2) to a concentration of ~1.0 x 10
8
CFU.mL
-1
(optical density at 600nm wavelength (OD
600
): 0.2). Thereafter,
100 μL of this suspension was inoculated into duplicates of 10 mL sterilized seawater (32 ppt) and incubated
