Identification of Cultivable Bacteria from Tropical Marine Sponges and Their Biotechnological Potentials.

Marine sponges are acknowledged as bacterial hotspots in the oceanic biome. Aquatic bacteria are being investigated comprehensively for bioactive complexes and secondary metabolites. Cultivable bacteria associated with different species of sea sponges in South China Sea waters adjacent to Bidong Island, Terengganu were identified. Molecular identification was accomplished using 16S rRNA gene cloning and sequencing. Fourteen bacterial species were identified and their phylogenetic relationships were analysed by constructing a neighbour-joining tree with Molecular Evolutionary Genetics Analysis 6. The identified species encompassed four bacterial classes that were Firmicutes, Actinobacteria, Alphaproteobacteria and Gammaproteobacteria known to have been associated with sponges. The potential biotechnological applications of the identified bacteria were compared and reviewed based on relevant past studies. The biotechnological functions of the 14 cultivable isolates have been previously reported, hence reinforcing that bacteria associated with sponges are an abundant resource of scientifically essential compounds. Resilience of psychrotolerant bacteria, Psychrobacter celer, in warm tropical waters holds notable prospects for future research.

Progressively known as a wealthy source of significant bioactive compounds, bacteria associated with sea sponges are receiving attention from the scientific community for novel secondary metabolites with desirable economical, pharmaceutical and cosmetic values (Thomas ). In this study, we identified 14 cultivable bacterial species related with sea sponges from adjacent waters of Bidong Island, Terengganu. Three different marine sponges namely Xestospongia sp., Haliclona sp. and Aaptos sp. were collected and identified. Bacteria associated with these sponges were identified and their biotechnological functions were addressed based on literature and previous researches.

A portion of the sponge surface (approximately 3 cm2 × 0.3 cm depth), which included the pinacoderm and mesohyl, was sampled using a sterile scalpel. The sponge tissue was divided into smaller pieces (2 mm3) using a sterile scalpel. Each 2 mm3 fragment was transferred into an Eppendorf tube. The sponge tissue fragments were washed with 1 mL of 0.22 μm filtered sterile natural seawater by gently shaking for 1 min at room temperature (25 ± 1°C), to remove loosely attached bacteria (Saidin ). The bacterial isolates went through primary screening based on colonial morphology observation on Zobell marine agar 2216 and Gram (1884) staining. The strains were isolated from the tissues of two Xestospongia sp., two Haliclona sp., and one Aaptos sp. marine sponges. The 16S ribosomal ribonucleic acid (16S rRNA) gene was amplified via direct colony polymerase chain reaction (PCR) by adding magnesium chloride (MgCl2), deoxyribonucleotide triphosphate (dNTP) mix, PCR buffer, forward primer 63F (5′-CAGGCCTAACACATGCAAGTC-3′) and reverse primer 1389R (5′-ACGGGCGGTGTGTACAAG-3′), Taq polymerase, and sterile distilled water into each tube. A single colony as template was added into each tube using sterilised toothpick (Riviere ). The Applied Biosystems® Veriti® 96-Well Thermal Cycler (Thermo Fisher Scientific Corp., California, United States) was set with pre-denaturation at 95°C for 5 min, followed by 26 cycles of denaturation, annealing and extension at 95°C for 1 min, 50°C for 30 s, and 70°C for 90 s respectively before proceeding with final extension at 72°C for 5 min (Turner ).

Subsequently, an agarose gel was prepared by dissolving 0.7% agarose powder in 1x tris-acetate-EDTA (TAE) buffer. The DNA ladder and PCR products with 6X loading dye were loaded into the wells. The loaded gel was run in an electrophoresis machine at a voltage, current and time of 90 V, 35 mA and 75 min respectively using Bio-Rad PowerPac™ Basic Power Supply gel electrophoresis machine (Bio-Rad Laboratories Inc., California, United States) (Sambrook & Russell 2006). The fluorescent Promega Diamond™ Nucleic Acid Dye was used to stain and view the gel using a Gel Doc XR+ Imaging System, which helped to determine the sizes of PCR DNA fragments.

After purification with Promega Wizard® Genomic DNA Purification Kit (Catalog No. A1120, Promega Corp., Wisconsin, United States), the PCR DNA was sequenced at First BASE Laboratories Sdn. Bhd. The DNA sequence for each isolate was identified using Standard Nucleotide BLAST program (BLASTN) to search the nucleotide databases using a nucleotide query, which is one of the programs under the BLAST® (Basic Local Alignment Search Tool, National Library of Medicine, Maryland, USA) program. The sequences were aligned using ClustalW sequence alignment program. A phylogenetic tree of the identified bacteria and their bacterial evolutionary relationships was inferred via Molecular Evolutionary Genetics Analysis version 6 (MEGA6) (Tamura ).

Fourteen bacteria species were identified by closest similarity with sequence databases using BLAST (Fig. 1). The identified sponge-associated bacteria species were Alteromonas macleodii, Bacillus aquimaris, B. aryabhattai, B. cereus, Brevibacterium epidermidis, B. iodinum, Enterobacter cloacae, Exiguobacterium indicum, Kytococcus sedentarius, Pseudomonas putida, Pseudovibrio denitrificans, Psychrobacter celer, Ruegeria arenilitoris, Staphylococcus hominis subsp. novobiosepticus. The phylogenetic tree in Figure 1 was inferred using the Neighbour-Joining method (Saitou & Nei 1987) with software MEGA6. The percentages of replicate trees where the associated taxa clustered together in the bootstrap test (10,000 replicates) are shown above the branches (Felsenstein 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura ) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. The phylogenetic tree analysed 15 nucleotide sequences: an ingroup comprising of 14 identified bacteria species and an outgroup of one microalga species.

Figure 1

Phylogenetic tree with 14 species ingroup and one microalga outgroup.

Fig. 1 shows a phylogenetic tree with the identified bacteria categorised into four bacteria groups, which are the phylum Firmicutes as well as the classes Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria. Bacteria from these similar groups have been previously isolated from marine sponges in geographically disparate areas such as the coastal waters of India, China, Ireland and Brazil (Webster & Taylor 2012). The previously reported geographic and biological isolation sources of these bacteria are shown in Table 1. Majority of species identified in this study were previously isolated from marine sponges, suggesting they were conceivably resident bacteria and not temporarily trapped transient bacteria (Table 1). Out of the 14 identified species, eight were ubiquitously present in more than one ocean and were also isolated from other marine invertebrates, they are Alteromonas macleodii (Mehta ), Bacillus aquimaris (Phelan , Cerritos ), B. aryabhattai (Pindi ; Paul ), B. cereus (Maliji ; Irshad ), Enterobacter cloacae (Maleki-Ravasan ; Altug ), Exiguobacterium indicum (Jimenez ; Pindi ), Pseudomonas putida (Altug ; Irshad ), and Pseudovibrio denitrificans (Santos ; Rua ). Ubiquitous bacteria are able to compete against exclusive sponge-associated bacteria for survival in marine hosts due to their flexibility in adapting to diverse environments, namely by exploiting resources, producing secondary metabolites and bioactive compounds, as well as tolerating broad ranges of temperature and salinity (Ivars-Martinez ). Consequently, the presence of competent ubiquitous bacteria within sponge larvae promotes the survivability of the latter when both symbiont and host are dispersed by ocean current to new habitats lacking in optimum physical parameters or symbionts (Thacker & Freeman 2012). As shown in Table 1, 8 out of 14 identified species were previously reported to have been associated with sponge hosts. A thorough search of relevant literature yielded no previous records that associated sea sponges with the remaining six identified species, which are Brevibacterium epidermidis, Brevibacterium iodinum, Ruegeria arenilitoris, Kytococcus sedentarius, Exiguobacterium indicum, and Staphylococcus hominis subsp. novobiosepticus. The presence of human microflora in sponge tissues implied that bacteria from heavily populated proximal waters may have passed through the filter-feeding sponges as temporary transient bacteria during sample collection, since geographically restricted human microflora has low survival rate against competent bacterial associates of sponges. However, future research is needed to study the relationship between the residing bacteria and the indigenous symbiont community, since the genome complexity and metabolic capabilities of the latter may gradually reduce due to exclusively restricted living environments within sponge hosts (Thacker & Freeman 2012).

Table 1

Sponge hosts and geographic locations of the isolated bacteria species.

Isolated species	Sponge hosts	Geographic locations	References
Alteromonas macleodii	Callyspongia sp.	Hong Kong	Qian et al. (2006)
	Gelliodes carnosa	Xincun Harbour, Hainan	Li et al. (2011)
Bacillus aquimaris	Haliclona simulans	Galway, Ireland	Phelan et al. (2011)
	Gelliodes carnosa	Xincun Harbour, Hainan	Li et al. (2011)
Bacillus aryabhattai	Sarcotragus fasciculatus	Bay of Bengal, India	Pandey et al. (2013)
Bacillus cereus	Haliclona simulans	Galway, Ireland	Phelan et al. (2011)
	Callyspongia sp.	Hong Kong	Qian et al. (2006)
	Hyattella cribriformis	India	Brammavidhya & Usharani (2013)
	Sigmadocia sp.	Kanyakumari coast	Satheesh et al. (2012)
	Spirastrella abata	Moseulpo Port, Jeju Island	Cho & Park (2009)
	Spirastrella panis	Moseulpo Port, Jeju Island	Cho & Park (2009)
	Polymastia janeirensis	Rio de Janeiro	Santos-Gandelman et al. (2014)
	Arenosclera brasiliensis	Rio de Janeiro	Rua et al. (2014)
	Stelletta tenui	Sanya, Hainan	Li et al. (2007)
	Dysidea avara	Sanya, Hainan	Li et al. (2007)
	Gelliodes carnosa	Xincun Harbour, Hainan	Li et al. (2011)
Enterobacter cloacae	Dysidea granulosa	Kavaratti Islands, India	Gopi et al. (2012)
Pseudomonas putida	Mycale microsigmatosa	Rio de Janeiro	Marinho et al. (2009)
Pseudovibrio denitrificans	Callyspongia plicifera	Bahamas	Qian et al. (2006)
	Spirastrella panis	Moseulpo Port, Jeju Island	Cho & Park (2009)
	Spirastrella abata	Moseulpo Port, Jeju Island	Cho & Park (2009)
	Arenosclera brasiliensis	Rio de Janeiro	Rua et al. (2014)
	Mycale microsigmatos	Rio de Janeiro	Santos et al. (2014)
Psychrobacter celer	Spirastrella abata	Moseulpo Port, Jeju Island	Cho & Park (2009)

Table 2 displays the biotechnological applications or potentials recorded for the species recognised in this study. Some were studied for the synthesis of enzymes and pharmaceutical or medical properties (Table 2). Among the identified species, the most common biotechnological function was associated with bioremediation, while subsequent common capabilities were plant growth promotion, plant systemic resistance, and biological syntheses of biocompatible nanometal and biodegradable polymers. We would like to note several interesting species that harbour unique abilities or products, namely lead, cadmium and zinc biosorption mechanism in A. macleodii (Loaëc ); halotolerant and psychrotolerant α-amylase with broad pH tolerance in A. macleodii (Han ); skeletal muscle relaxation properties and central nervous system (CNS) depressants in B. cereus (Kumar ); antileukaemic and antineoplastic L-asparaginase in B. aryabhattai (Singh & Srivastava 2014); polyhydroxyalkanoate (PHA) synthesis in P. putida (Wang & Nomura 2010); biodegradation of highly explosive pentaerythritol tetranitrate (PETN) by E. cloacae (Binks ); as well as antibacterial, immunosuppressive and anticancer prodigiosin in P. denitrificans (Sertan-de Guzman ).

Table 2

Review of potential biotechnological applications of the isolated bacteria species.

Isolated species	Biotechnological application	References
Alteromonas macleodii	Metallic ion biosorption	Loaëc et al. (1997)
	Hydrogenase enzyme production	Vargas et al. (2011)
	α-amylase enzyme production	Han et al. (2014)
	Biocompatible nanometal production	Mehta et al. (2014)
	Alkaline phosphatase production	Fedosov et al. (1991)
Bacillus aquimaris	Alkaline cellulase production	Trivedi et al. (2011)
	α-amylase enzyme production	Puspasari et al. (2013)
Bacillus aryabhattai	β-glucosidase inhibitor production	Pandey et al. (2013)
	L-asparaginase enzyme production	Singh & Srivastava (2014)
	Polluted water bioremediation	Chen et al. (2012)
	Bagasse degradation	Wen et al. (2015)
Bacillus cereus	Antimicrobial	Brammavidhya & Usharani (2013)
	Therapeutic depressant potential	Kumar et al. (2014)
	Petroleum hydrocarbon degradation	Maliji et al. (2013)
	Mercury bioremediation	Santos-Gandelman et al. (2014)
	Silver nanoparticle production	Babu et al. (2011)
Brevibacterium iodinum	Plant germination and growth promotion	Kim et al. (2012)
	Iodinin antibiotic production	Podojil & Gerber (1967)
Enterobacter cloacae	Greenhouse cucumber growth regulator	Georgieva (2003)
	Explosives biodegradation	Binks et al. (1996)
Exiguobacterium indicum	Wastewater bioaugmentation	Iyer & Iken (2013)
	Manganese mobilisation	Sujith et al. (2014)
Kytococcus sedentarius	Keratin-degrading enzyme production	Longshaw et al. (2002)
	Monensin antibiotic production	Pospisil et al. (1998)
Pseudomonas putida	Antimicrobial	Marinho et al. (2009)
	Polyhydroxyalkanoates (PHA) production	Wang & Nomura (2010)
	Plant systemic resistance induction	Planchamp et al. (2015)
	Polluted water bioremediation	Shimazu et al. (2003)
	Certified host-vector biosafety strain (HV1)	Federal Register (1982)
Pseudovibrio denitrificans	Pseudovibrocin peptide production	Vizcaino et al. (2010)
	Prodigiosin pigment production	Sertan-de Guzman et al. (2007)

The commonly recorded capability of bioremediation in some of the identified ubiquitous bacteria is explicable, for the degradation and utilisation of surrounding resource allowed these adaptable bacteria to tolerate severe environments by exploiting nutrients, producing secondary metabolites and bioactive compounds, and tolerating broad ranges of temperature and salinity (Ivars-Martinez ), hence eventually settling as competent symbionts of marine invertebrates. Moreover, it should be brought to attention that the water bioremediation capacity of Pseudomonas putida (Table 2) was not an innate ability but expressed via ice-nucleation protein (INP) surface anchor, which revealed a ten-fold catalytic reaction compared to preceding research on Escherichia coli (Shimazu ). Additional common biotechnological aptitude was the biosynthesis of nanometals that has been earlier reported for two identified species in this study, which are A. macleodii and B. cereus. Bacteria that biosynthesise nanometal particles are resistant to heavy metals, which could be found in ports and waters polluted with heavy metal effluents (Ramanathan ).

A lack of previous reports were available for the biotechnological applications of B. epidermidis, R. arenilitoris, P. celer, and S. hominis subsp. novobiosepticus. Among the four strains, B. epidermidis is characteristically found on human skin as an ordinary human microflora. Although research on B. epidermidis is deficient, human microflora may harbour unknown dermatological prospects, such as callous treatment discovered in a human microflora recognised as Kytococcus sedentarius (Longshaw ). Furthermore, P. celer is of a psychrophilic or psychrotolerant genus, Psychrobacter sp., whereby its ability to thrive in both warm and cold regions may be a desirable trait for industrial research purposes (Rodrigues ). R. arenilitoris is a relatively recent species requiring research on its biotechnological prospects (Park & Yoon 2012). Although the recognised species were chiefly ubiquitous bacteria whereby they were neither exclusive nor specific to sea sponges, the results of this research nevertheless correspond with past studies that sponges are abundant with microbes capable of pharmaceutical and biotechnological applications (Schippers ).

The biotechnological uses shown in Table 2 show ample research prospects among the identified cultivable symbionts associated with sponges. However, we would like to note that the same bacteria species does not necessarily yield similar products due to the mutation or adaptation of strains in respect to their surrounding environment. Majority of the identified species were ubiquitous with proficient survival mechanisms that promote adaptability in harsh environments, thus opportunistically outgrowing adjacent bacteria in marine sponges and residing as a part of the symbiont community in marine sponges. Furthermore, ten identified species were recorded in past studies to have harboured at least two biotechnological purposes, illuminating sponges as a markedly wealthy mine of microbes that are medically and biotechnologically substantial. However, cultivable bacteria under laboratory conditions occupy less than 1% of the total sponge microbial community (Amann ), suggesting an uncharted existence of valuable research prospects within the putative 99% of uncultivated sponge bacteria. As a result, research is essential on growth media that foster the development of sponge symbionts, and on progressive metagenomic approaches that access the environmental metagenome devoid of isolation on complex culture media.