Background

Cellulose is composed of glucose polymers joined together by β-1, 4 glycosidic linkages (O'sullivan, 1997). Due to the high glucose content and as the world’s most abundant organic materials (Siró and Plackett, 2010), cellulose is considered as rich and cheap energy sources for some terrestrial animals which are able to produce cellulase endogenously, a group of enzyme which could break down the β-1, 4 glycosidic linkages and produces glucose units (Spano et al., 1976). However, several aquatic species are unable to directly utilized cellulose, because they are unable to produce the cellulase enzyme (Bondi and Spandorf, 1954; Fish, 1960; Stickney and Shumway, 1974; Li et al., 2009). Therefore, cellulose content in aquaculture diets is frequently associated with adverse effects on cultivated species (Li et al., 2009). For instance, the use of high cellulose food ingredients such as soybean meal or microalgae as a replacement of fish meal has been observed to decrease food digestibility (Appler, 1985; Francis et al., 2001). In addition, high cellulose content in aquaculture feed has been described to increase intestinal viscosity and reduce digestive-enzyme access to other food ingredients, resulted in lower feed intake and low growth of aquaculture animals (Francis et al., 2001). It was generally accepted that cellulose could only be utilized by several terrestrial animals only such as termite (Ohkuma, 2003) and ruminants (Stewart, 1977). However, a study by Davies (1965) discovered that some bacteria associated with intestinal tracts of cellulose - utilizing animals, were able to produce cellulase and played significant roles in cellulose degradation. Since then, numerous studies have been conducted to isolate and investigate cellulose-degrading bacteria for diverse purposes.

The inclusion of cellulose - degrading bacteria has been documented to increase cellulose digestibility and yield more metabolizable energy in ruminants (Varel, 1987; Titi and Tabbaa, 2004). The use of beneficial bacteria to enhance performance of animals is generally known as probiosis. The same approach can be practiced in aquaculture species especially to aquatic species with lack of cellulolytic activity. The supplementation of cellulose-degrading bacteria may eliminate the adverse effects of cellulose content in plant-based diets and improve food digestibility as well as the growth of cultivated species. Verschuere et al. (2000) described that probiotic bacteria should be viable in order to give significant contribution to animal hosts. Therefore, Fjellheim et al. (2010) suggested that probiotic bacteria for aquaculture species should be isolated from aquaculture animals. These bacteria are presumed to be well adapted to the targeted ecological niche and may have greater chance of colonizing their new host and contribute in food digestions.

Thus, this study aimed at isolation and characterization of bacteria with cellulolytic activity from the gastrointestinal tract of hybrid abalone as probiotic candidates. In addition; the capacity of cellulose - degrading bacteria to tolerate seawater and to adapt to the gastrointestinal tract environment of aquatic species was further investigated. The result of this study is expected to give a contribution to develop cheaper diet ingredients such as plant - based diets as alternative replacement of high-priced fish meal in formulated feed.

1 Result

1.1 Cellulose - degrading bacteria

A total of 15 bacterial isolates were initially isolated and purified from the enrichment culture method using the CMC broth medium. Of these, only 7 isolates were confirmed to have cellulolytic activity, indicated by a discoloration of Congo - red agar (Figure 1). Phenotypic studies showed that all isolates were catalase positive, and under microscope appeared to be rod. Most isolates were gram positive (6/7) and (4/7) were able to produce oxidase (Table 1).

1.2 Cellulolytic activity and bacterial identification

Quantitative assay indicated that there was a significant difference in the amount of reducing sugar among the seven bacterial isolates, F = 86.37; df 7, 10; p = < 0.01. In general, the highest amount of reducing sugar was observed from cell - free supernatant collected from isolate C-aw2 followed by C -1 (Figure 2). Based on partial sequence of 16S rDNA, isolate C - aw2 showed 95% similarity to Stenotrophomonas sp strain LY -2 (acc. nb. LC136 883), and isolate C -1 showed high similarity (99%) to Bacillus sp strain WR -2 (acc. nb. KU159 243).

1.3 Survival in seawater

Both Stenotrophomonas sp and Bacillus sp had high survival rate after 6 h exposure in seawater (32 ppt) (Figure 3). In fact, Bacillus sp was able to grow in the seawater, indicated by the increasing number of viable cells after the 6 h exposure, from 6.15 at 0 h to 6.67 Log (CFU. mL^-1) after 6 h, t =13.3, df 1, p =0.006. Meanwhile, there was no significant difference in the average number of viable cells for Stenotrophomonas sp at 0 h and after 6 h exposure into seawater, t =1.74, df 1, p = 0.33.

1.4 Survival in simulated stomach condition

Both bacterial isolates showed high survival rate in the simulated stomach environment (Figure 4), indicated by no significant difference in the number of viable cells (Log CFU.mL^-1) at 0 h and after 3 h incubation. Viable cells of Stenopthromonas sp were recorded to be 6.04 at 0 h and became 6.03 after 3 h incubation, which was no statistically different, t= 0.33, df 1, p =0.79. Meanwhile, the number of viable cells of Bacillus sp was significantly higher at 3 h (7.12) after exposure compared to 0 h (6.54), which may also indicate the capacity of this bacterial isolate to grow in the simulated stomach environment, t =206.9, df 1, p < 0.01.

1.5 Survival in simulated intestinal condition

There were no significant differences in the viable cells of both cellulose-degrading bacteria (Log. CFU.mL^-1) at 0 h and 4 h, (t=3.46, df 1, p=0.18 for Bacillus sp; and t=5.60, df 1, p=0.11 for Stenotrophomonas sp). In general, viable cells of Stenotrophomonas sp and Bacillus sp were slightly higher, but statistically no significant difference after the 4 h incubation in the intestinal simulation (Figure 5).

1.6 Toxicity

Both Stenotrophomonas sp and Bacillus sp were found to be harmless towards juvenile hybrid abalone, indicated by no mortality or sign of diseases were observed during the 14-day feeding trial.

1.7 Phylogenetic tree

A phylogenetic tree was constructed based on partial sequence of 16S rRNA gene and GeneBank data-base reference sequence using Genious software version 5.3.6 (Figure 6). The result revealed that isolate C - aw2 was very close to Stenotrophomonas sp LY -2 and isolate C -1 indicated to have a closed relatedness with Bacillus sp. strain WR -2.

2 Discussions

Plant-based proteins such as soybean meal, and microalgae has been considered as an alternative replacement for fish meal in the commercial aquaculture feed (Appler, 1985, Gomes et al., 1995 ; Kaushik et al., 1995). However, several studies indicate that the use of plant-based diets generally display lower food digestibility than animal-based diets. And this is caused mainly by the presence of cellulose (Boyd and Goodyear, 1971), which is main membrane structure surrounding cell wall of soybean meal and of the aquatic plant (Baldan et al., 2001 ; Mihranyan, 2011). In addition, cellulose content in the plant-based aquaculture feed has been frequently associated with several adverse effects on digestive processes, including : increasing in viscosity of intestinal juice, reducing an access of digestive enzyme to other feed materials (Bromley and Adkins, 1984), and lowering food intake which results in slowing the growth of cultured animals (Francis et al., 2001). One way to break down cellulose content in the plant-based diets is by the use of cellulose-degrading bacteria. Therefore, this study was performed in the effort to find novel strains of intestinal bacteria with the capacity of degrading cellulose.

The result showed that there were 7 endosymbiont bacteria exhibiting capacity to degrade cellulose. To the author’ knowledge, this is the first study to report cellulose - degrading bacteria from hybrid abalone. The number of cellulose-degrading bacteria detected in the animal gut might not represent the correct pictures of cellulose-degrading bacteria, regarding the isolation method used in this study (Ellis et al., 2003). More advanced techniques such as polymerase chain reaction - denaturating gradient gel electrophoresis (Muyzer and Smalla, 1998) or other culture - independent metagenomic approaches (Nakamura et al., 2016) might give more precise number of cellulose-degrading species. However, as the main concern of this study is not only detecting the presence of cellulolytic bacteria, but also investigating their potential capacity as aquaculture probionts, the use of cultured-dependent technique is considered legitimate.

Of these 7 cellulose - degrading bacteria, two baceterial isolates exhibiting the highest 2 cellulolytic activity (Stenotrophomonas sp and Bacillus sp) were further investigated for their probiotic properties including: adaptability to seawater and simulated gastrointestinal tract of aquatic species, as well as safety of these bacteria to cultured animals which in fact are among the most critical factors for selecting probiotic candidates (Verschuere et al., 2000; Merrifield et al., 2010; Geraylou et al., 2014). The results showed that both bacterial strains are resistance to high salinity (32 ppt) in seawater, low pH (4.5) which is much lower compared to pH of abalone stomach (5.45) (Harris et al., 1998) and bile salt (0.3%) in the intestine. This result could be initial indicators for the capacity of these cellulose-degrading bacteria to colonize intestinal tract, acknowledging some bacterial sensitivity to low pH in stomach and bile salt in intestine (Giannella et al., 1972; Borriello et al., 1985). In fact, Bacillus sp seemed able to grow in the seawater and simulated stomach juice. Regarding the capacity to tolerate bile salt, these bacteria might produce an enzyme which break down bile salt called bile salt hydrolase (Du Toit et al., 1998) or due to the production of protective coating of exopolysaccharide (Roberts and Powell, 2005). These results may suggest that both bacteria are capable to tolerate and grow in intestinal tract of aquatic species and contribute to food digestions. In addition, as safety for the animal host is another primary requisite of any probiotic bacterium (Verschuere et al., 2000), both bacterial strains had been confirmed to be harmless to the juvenile of hybrid abalone. During 14 - day feeding trials, there were no disease signs or mortalities observed from the juvenile abalone. These results may suggest that these cellulose-degrading bacteria are potential candidate for aquaculture probionts.

The supplementation of cellulose - degrading bacteria into cultured animals can be considered as an alternative to enhance digestibility processes of culture animals. For instance in larval stages, many aquatic species which rely mostly on cellulose-rich diets such as microalgae (Reitan et al., 1997) have been reported to have poor digestibility in microalgae (Skrede et al., 2011). The poor digestibility has been associated with the thick and rigid algal cell wall (Becker, 2007; Marshall et al., 2010). As the cell wall is also composed by cellulose, these types of bacteria may be used to help breaking down the algal - cell walls (Mihranyan, 2011). In addition, the inclusion of 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 KH₂PO₄, 0.25 g. L^-1 MgSO₄, 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 of15 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^oC. 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^oC) for 7 days. Cells - free supernatant was harvested by centrifugation at 5 000 rpm for 15 min at 4^oC. 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 10⁸ CFU.mL^-1 (optical density at 600nm wavelength (OD₆₀₀): 0.2). Thereafter, 100 μL of this suspension was inoculated into duplicates of 10 mL sterilized seawater (32 ppt) and incubated aerobically at 21^oC for 6 h. The viability of bacterial isolates was evaluated by pipetting 100 µL of the mixture, serially diluted and plated onto CMC agar at 0 h and 6 h. After 2- day incubation, the number of bacterial colonies on each plate was enumerated.

3.5 Survival in stomach environment

A simulated stomach juice was prepared according to a modified protocol of Geraylou et al. (2014). In brief, pepsin (3 mg. mL^-1) was diluted in normal saline solution (NSS; 0.85%), and pH was adjusted to 4.53 with 1 N hydrochloric acids (HCL). Fresh culture of cellulose - degrading bacteria was harvested by centrifugation, and the bacterial cells was washed and suspended into PBS (~ 1.0 x 10⁸ CFU.mL^-1). Thereafter, 100 μL of this suspension was inoculated into duplicate of 10 mL stomach simulation, and incubated for 3 h at 21ºC. Then, viable cells of the tested bacteria were monitored by plating the bacterial mixture onto CMC - agar at 0 h and after 3 h incubation.

3.6 Survival in intestinal condition

A simulated intestinal juice was prepared by mixing these chemical compounds (125 m.mol^-1 NaCl, 7 m.mol^-1 KCl, 45 m.mol^-1 NaHCO³, 0.3% bile salt and 3 g.L^-1 trypsin) in sterile distilled water. Subsequently, 100 μL of fresh bacterial culture of cellulose – degrading bacteria (~ 1.0 x 10⁸ CFU.mL^-1) was inoculated into duplicates of 10 mL intestinal simulation; followed by incubation for 4 h at 21ºC. Then, cell viability was monitored by plating of the mixture on CMC agar at 0 h and after 4 h incubation.

3.7 Toxicity assay

A total of 30 juvenile hybrid abalones (0.47±0.13 g) were divided into 3 experimental groups in a small - scale in vivo experiment. Each group had 2 rearing tanks and each tank had 5 juvenile abalones. The abalone were fed with 1.5% body weight.day^-1 with commercial pellet impregnated with either Stenotrophomonas sp or Bacillus sp at a cell concentration of ~1.0 x 10⁹ CFU.g^-1. The animals were reared in 10 L filtered seawater (32 ppt) for 14 days, and the rearing water was replaced every three days. During the experiment, dead abalone was recorded on daily basis.

3.8 Data analysis

Data obtained were statistically analyzed using either independent Sample t - test or one-way analyses of variance (ANOVA), followed by tukey comparison test.

Acknowledgement

This work was supported by the Institute for Marine and Antarctic Studies (IMAS), an institute of the University of Tasmania. The author appreciates Nick Savva for providing abalone used for this study.

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