Abstract: Background and Objective: A new green technology to reduce environmental damages while optimizing production of Pacific Whiteleg shrimp, Litopenaeus vannamei was developed known as "Biofloc technology". Microbial communities in biofloc aggregates are responsible in eliminating water exchange and producing microbial proteins that can be used as supplemented feed for L. vannamei. This study aimed to isolate and identify potential bioflocculant-producing bacteria to be used as inoculum for rapid formation of biofloc. Materials and Methods: For the purpose of this study, bacterial communities during 0, 30 and 70 days of culture (DOC) of L. vannamei grow-out ponds were isolated and identified through phenotypic and 16S rDNA sequences analysis. Phylogenetic relationships between isolated bacteria were then evaluated through phylogenetic tree analysis. One-way analysis of variance (ANOVA) was used to compare the differences of microbial communities at each DOC. Results: Out of 125 bacterial isolates, nine species of bacteria from biofloc were identified successfully. Those bacteria species were identified as Halomonas venusta, H. aquamarina, Vibrio parahaemolyticus, Bacillus infantis, B. cereus, B. safensis, Providencia vermicola, Nitratireductor aquimarinus and Pseudoalteromonas sp., respectively. Through phylogenetic analysis, these isolates belong to Proteobacteria and Firmicutes families under the genera of Halomonas sp., Vibrio sp., Bacillus sp., Providencia sp., Nitratireductor sp. and Pseudoalteromonas sp. Conclusion: In this study, bioflocculant-producing bacteria were successfully identified which are perfect candidates in forming biofloc to reduce water pollution towards a sustainable aquaculture industry. Presence of Halomonas sp. and Bacillus sp. in all stages of biofloc formation reinforces the need for new development regarding the ability of these species to be used as inoculum in forming biofloc rapidly.
INTRODUCTION
Growing human population that increases demand for food supply has led to intensive development of aquaculture industry worldwide, particularly in Asia. However, development of aquaculture industry has brought negative effects to the environment and natural sources through pollution of ground and surface waters by effluent discharge1,2.
A new technology to reduce environmental damages and optimizing production on this industry has been developed known as "Biofloc technology" (BFT). The BFT is an efficient aquaculture system due to the ability of biofloc to continuously recycled and reused nutrients in the culture pond3. This is achieved by maintaining high ratio of carbon to nitrogen (C:N) in the water through addition of external carbon sources such as molasses or starch to stimulate heterotrophic bacterial growth that converts ammonia into microbial biomass4. The BFT are able to eliminate water exchange in aquaculture systems by maintaining optimum water quality as well as producing microbial protein that act as supplemented feed for aquatic organisms5.
"Biofloc" is composed of aggregates of microorganisms including bacteria, fungi, microalgae, protozoans and rotifers and other types of particulate organic matter such as faeces and unused feed6. Bacteria as main component of biofloc were reported as bioflocculant-producing microorganisms that produced biopolymer substances that can flocculate suspended solids, cells and colloidal solids7. These biopolymers known as Extracellular Polymeric Substance (EPS) produced by microorganisms during their growth play an important role in flocculation process8,9. However, there is a lack of basic knowledge regarding microbial composition in biofloc5,10. As bacteria play significant roles in biofloc formation, a clear understanding on microbiological aspects mainly in microbial communities in biofloc is important for the effective design and successful operation of biofloc technology. Therefore, this study aimed to isolate and identify bacteria from biofloc through 16S rDNA sequences analysis which later can be used as potential inoculum for rapid biofloc formation.
MATERIALS AND METHODS
Biofloc sample collection: Biofloc sampling was conducted at the Integrated Shrimp Aquaculture Park (iSHARP), Blue Archipelago at Setiu, Terengganu (Fig. 1). The biofloc samples were collected from selected L. vannamei ponds during 0, 30 and 70 days of culture (DOC) as at this time, biofloc was observed to be formed. Two litres of biofloc samples was collected in pre-acid washed plastic bottles (1 L) and was taken back to laboratory for further analysis. The samples were then transferred into Imhoff cones to enable the biofloc to settle at the bottom of the cone for 24 h.
Preparation of biofloc samples and isolation of bacteria: The settled biofloc samples from Imhoff cone were transferred into a centrifuge tube for centrifugation at 6000 rpm for 3 min. The biofloc pellets were collected and was streaked onto marine agar as the cultivation medium. The cultures were then incubated for 24 h. The colonies which appeared on plates were purified by repeated streaking and Gram staining was performed to ensure the purity of the colony.
Identification of isolated bacteria based on phenotypic characterization: To identify the isolated bacteria, Gram staining was carried out according to standard microbiological protocol. The colonies were distinguished through visual observations of colony morphology. Individual colonies were characterized through commonly used biochemical test.
Genetic characterization and diversity analysis: Single colony of pure culture bacteria was grown in marine nutrient broth for 16-18 h. Cultures were then centrifuge at 9000 rpm for 15 min and the supernatant was decanted. The DNA extraction for bacteria using the Qiagen DNeasy Blood and Tissue Kit was conducted as per manufacturer’s protocol.
Universal primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) described by Yu12 were used to amplify the bacterial 16S rDNA gene. Polymerase Chain Reaction (PCR) was performed using GoTaq® PCR Core Systems (Promega, USA). All PCR reagents were used following recommended reaction volumes and final concentrations provided by manufacturer. The PCR amplification was performed in VeritiTM Thermal Cycler (Applied Biosystems, USA). The reaction conditions included an initial denaturation at 94°C for 1 min, followed by 35 cycles in series of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 90 sec, with a final extension at 72°C for 7 min13. The amplification product were separated by electrophoresis on a 1% agarose gel and purified using QIAQuick PCR purification kit (Qiagen, USA) according to manufacturer’s protocol. The purified PCR products were sent to First Base Sdn. Bhd. for sequencing process.
| Fig. 1: | Location of the L. vannamei culture ponds under the management of iSHARP, Blue Archipelago Sdn. Bhd. at Setiu District, Terengganu, Malaysia11 |
Homologies of query sequences were searched at the National Centre for Biotechnology Information (NCBI) GenBank nucleotide database using Basic Local Alignment Search Tool (BLAST) through website14. The sequences were further subjected to Multiple Sequence Alignment (MSA) using ClustalX15. A phylogenetic tree using 16S rDNA sequences of isolated bacteria was constructed using Mega6 software (Version 6.06)16.
Statistical analysis: All data were tested for homogeneity (O’Brien, Brown-Forsythe, Levene and Bartlett tests) and normality (Shapiro-Wilk test). When variances of these data were equal, parametric t-tests were performed. Differences of microbial communities with DOC were compared using one-way analysis of variance (ANOVA)17. One-way ANOVA was used because there was only one independent variable in this study which was number of bacterial species in each DOC. Statistically significant differences were accepted with α of 0.05. All statistical analysis was performed using JMP-IN (Version 4.0.3, S.A.S Institute Inc. Cary, USA).
RESULTS
A total of 10 bacterial isolates marked as SP1-SP10 were isolated from L. vannamei culture ponds. From these ten species, nine species were isolated from biofloc samples of L. vannamei pond. The colony characteristics of all isolated bacteria were analyzed. All colonies appeared as white to creamy in colour. The isolated bacteria observed to form mostly circular and smooth colonies on agar surfaces. All bacteria from phylum firmicutes were determined as Gram positive and spore forming while bacteria from phylum Proteobacteria were determined as Gram negative and non-spore forming.
To support this morphological and microscopic study, taxonomical identification was further investigated through molecular study and phylogenetic analysis. The 16S rDNA gene of 125 bacteria isolates of approximately 1500 bp were successfully amplified by PCR (Fig. 2). The 16S rDNA sequence data were subjected to a BLASTn search. The homology search results of bacterial strains resembling with existing DNA sequence database were identified (Table 1).
Bacteria SP1-SP7 were found to belong to group Proteobacteria showing 99-100% with Halomonas sp., Vibrio sp., Nitratireductor sp., Alteromonas sp., Providencia sp. and Pseudoalteromonas sp. genera, respectively. The analysis using BLASTn tool showed SP8, SP9 and SP10 belonged to Firmicutes branch showing sequence similarity of 98-100% with genera Bacillus sp. Phylogenetic tree also supported the blast analysis report and showed separate line of descent in the Proteobacteria and Firmicutes (Fig. 3).
In this study, biofloc was observed started to be formed in 30 DOC up to 70 DOC. The 0 DOC of shrimp culture showed the lowest number of bacteria species isolated with seven species while 30 DOC showed the highest species isolated with nine species (Fig. 4). The results showed that there was significant difference (p<0.05) of bacterial species in biofloc samples between DOC of L. vannamei culture periods.
| Fig. 2: | Electrophoresis gel stained by ethidium bromide of 16S rDNA gene of PCR product using 1492R and 27F primers |
| Fig. 3: | Neighbour-joining phylogenetic tree of 10 bacterial species based on 16S rDNA sequencing depicting homology to closely related bacterial species |
| Note that bootstrap support values over 60% are shown. The scale bar indicates evolutionary distance | |
DISCUSSION
The findings of this study successfully provided substantial evidence that microbial composition in biofloc had enhanced water quality condition of L. vannamei culture. Microbial composition in 0 DOC showed the least bacteria species due to low concentration of organic matter and carbon sources where stocking of L. vannamei was not yet been introduced into the culture pond.
| Fig. 4: | Number of bacterial species isolated from biofloc of L. vannamei culture grow-out pond |
| Error bars represent standard errors | |
| Table 1: | List of bacteria isolates identified by 16S rDNA analysis deposited in NCBI, GenBank, USA with accession numbers |
As there was no biofloc was formed in 0 DOC, thus only bacteria that naturally present in the L. vannamei culture pond were isolated. Bacteria genera such as Bacillus sp., Halomonas sp., Vibrio sp., Nitratireductor sp., Alteromonas sp. and Pseudoalteromonas sp. that were successfully isolated and identified at 0 DOC were bacteria species that can be found in marine water18-22. Bacteria act as an efficient "Biochemical systems" degrader and metabolize organic residues23. In other words, they recycled nutrients efficiently in a form of organic and inorganic matter (unconsumed and non-digested feed, metabolic residues and carbon sources applied as fertilizers) into new microbial cells. Microorganisms that populate biofloc systems typically inhibit the natural aquatic systems and highly influenced by factors such as light intensity and concentration of organic matter24. This was proven where the presences of bacteria species during DOC 0 were also found in biofloc during 30 DOC.
The highest number isolated bacteria species from biofloc was at 30 DOC. The introduction of commercial shrimp feed that contains new bacteria species might have contributed to additional of four new bacteria species. For example, bacteria that were absent in BWT but exist in 30 DOC such as H. aquamarina, Providencia sp. and B. cereus. The presence of bacteria in commercial shrimp feed was partially depends on the ingredients used in feed formulation25. In this study, shrimp feed used were made from raw materials such as wheat flour, soybean, squid, fish meal and yeast. These raw materials were often contaminated with bacteria such as Bacillus sp. and Vibrio sp.25. On the other hand, bacteria species such as Halomonas sp. and Providencia sp. were reported as marine bacteria and can be found in marine environment26.
Presence of bacteria genera of Pseudoalteromonas sp., Bacillus sp. and Halomonas sp. are beneficial for growth of shrimp culture as well as maintaining water quality of shrimp pond. Pseudoalteromonas sp. and Halomonas sp. have been used as probiotics in shrimps culture27,28 while addition of Bacillus sp. has contributed in maintaining water quality by reducing total suspended solids in shrimp ponds28. In contrast, isolation of Vibrio sp. in shrimp pond and biofloc samples did not causing any outbreak of disease. Although Vibrio sp. has been reported to cause diseases to shrimps, it was concluded that all Vibrio species was differ in virulence and must be present at certain threshold for disease to occur29. Acceptable level for total number of V. parahaemolyticus was below than 100 CFU mL–1 as reported by Shaari et al.30. In addition, presence of Halomonas sp. and Bacillus sp. has been found to have an inhibitory effect in vitro against V. parahaemolyticus and V. harveyi with antagonistic substances excretion31. This might improve immune system of L. vannamei as well as shrimp’s resistance towards pathogen V. alginolyticus32,33.
Interestingly, Bacillus sp. and Halomonas sp. were isolated in all DOC either from pond water or biofloc sample. Even though both genuses were dominantly exist in marine environment34, both species have been reported to be a bioflocculant-producing microorganisms34-36. Bacillus sp. was reported to have a high flocculating activity of more than 90% and capable to produce polysaccharide bioflocculant36 whereas Halomonas sp. was found to have flocculating activity of 95% and producing mainly polysaccharide bioflocculant37,38.
The findings of this research was successfully provide substantial evidence as composition of heterotrophic bacteria which has the ability to produce bioflocculant are perfect candidate in forming biofloc which later can reduce water pollution. Up to date, there is a limited study on determination of microbial communities within biofloc. The uniqueness and biodegradability of microbial flocculants have prompted research into screening, characterization and structural identification of polymeric flocculants excreted by the microbes. The paucity of information on those aspects and further research is needed in order to develop better and environmentally safer alternatives as compared to the synthetic flocculants. Thus, those identified bacteria are highly potential to be used as inoculum in forming biofloc for sustainable aquaculture industry.
CONCLUSION
Nine species of bioflocculant-producing bacteria known as Halomonas venusta, H. aquamarina, Vibrio parahaemolyticus, Bacillus infantis, B. cereus, B. safensis, Nitratireductor aquimarinus, Providencia vermicola and Pseudoalteromonas sp. from biofloc of shrimp pond were successfully identified through 16S rDNA sequences analysis. Existence of heterotrophic bacteria genera such as Halomonas sp. and Bacillus sp., in biofloc showed high potential to be used as inoculums for rapid formation of biofloc towards sustainable aquaculture practices.
SIGNIFICANCE STATEMENTS
The findings of this study will benefit to the shrimp farmers around the world in implementation of biofloc technology that will minimize water exchange and enhance the growth of shrimp culture. The addition of isolated bioflocculant-producing bacteria as inoculum plays an important role to boost-up the formation of biofloc. Therefore, this sustainable aquaculture approach will maximize the overall production of shrimp to meet the market demands, while preserving environmental safety.
ACKNOWLEDGMENTS
This project was financially supported by the Ministry of Education, Malaysia (MOE) under Fundamental Research Grant Scheme, FRGS (Grant number: 59401). We also would like to thank iSHARP, Blue Archipelago Berhad at Setiu, Terengganu, Malaysia for L. vannamei aquaculture facilities. Finally, to all lab staffs at Institute of Tropical Aquaculture (AKUATROP), Universiti Malaysia Terengganu who have major contributions throughout the study periods.