Biofouling is the accumulation of unwanted biological matter on surfaces. Microorganisms produce biofilms and macroorganisms produce macroscale biofouling (Bhushan 2016). It causes harmful environmental effects due to increased harmful emissions and loss of biodiversity (Subba Rao 2005). The process of biofouling occurs as a sequence, beginning with biofilm-forming organisms attaching and initially colonising surfaces; invertebrates are then attracted and their subsequent attachment to the hull's surface is supported (Vinagre et al. 2020). Antifouling systems using antifouling paint coatings and other means have been used for decades to overcome biofouling in the maritime sector (Yebra et al. 2004). One of the most effective and successful paints commonly used to prevent biofouling is a self-polishing copolymer paint containing the biocide tributyltin (TBT). However, its application has a negative effect due to its extreme toxicity towards non-target organisms, such as reproduction failure and shell deformity in molluscs; bivalves and gastropods are especially sensitive to TBT (Alzieu 1998). Its use was therefore banned by the International Maritime Organization in 2003, leading to the need for TBT-free paints in antifouling applications (Chambers et al. 2006). To ensure environmental sustainability in the treatment of biofouling, TBT and other organotin-based paints need to be replaced by antifouling compounds that are highly effective, yet less toxic to the environment (Qian et al. 2009).
Natural products have been used as new, effective and nontoxic sources of antifouling compounds to replace TBT-based paints and other toxic products (Wang et al. 2017). Natural products from marine organisms are good candidates for antifouling due to the organisms’ ability to survive biofouling through various strategies. The secondary metabolites they produce have bioactive properties that prevent predation and colonisation of their surfaces by fouling organisms (Wahl et al. 2014). Most of the secondary metabolites produced by marine organisms have inhibitory activity against biofouling organisms, which makes them a source of antifouling compounds (Carroll et al. 2021). Soft corals and macroalgae can resist colonisation of their surfaces by fouling organisms. Surface-associated marine bacteria are a unique source of new compounds with diverse chemical structures and bioactive properties, including antifouling (Wang et al. 2022). They are currently being explored as new sources of antifouling compounds due to their broad spectrum of activity (Gomez-Banderas 2022). They are considered to be the source of the next generation of bioactive compounds in the near future due to the many effective new compounds that are being discovered in them in different studies around the world, as reviewed recently (Srinivasan et al. 2021). Many diverse natural products of these organisms have been reported, and many more chemically unique compounds are being discovered (Blockley et al. 2017). The bacteria perform several roles necessary for the survival of their host, such as providing nutrients like carbon and nitrogen (van de Water et al. 2018) and chemical defences that protect the host against colonisation and predation (Krug 2006; Kamat et al. 2022). Potent antifouling compounds have been isolated from the bacteria associated with the living surfaces of marine organisms such as soft corals and macroalgae (Satheesh et al. 2016). Many compounds from the bacteria found in association with these organisms have been reported to have antifouling activity (Holmström et al. 2002; Ma et al. 2009).
Soft coral-associated bacteria have been described as a rich source of bioactive compounds with antifouling activity (Modolon et al. 2020). Macroalgae-associated bacteria are another important source of bioactive compounds, with several bacteria having been reported to show antifouling activity (Egan et al. 2001). Tackling biofouling will require compounds with multiple activities against the different group of organisms that attach and colonise on the surface to form the fouling structure. The compounds should have an inhibitory effect on bacterial growth, biofilm formation and larval settlement. To date, only a few studies have evaluated compounds for multiple inhibitory activity, especially antisettlement activity. In this study, surface-associated bacteria from the soft corals and macroalgae from the Red Sea were evaluated for antibiofilm activity against biofilm-forming bacteria and antisettlement activity against barnacle larvae. A study of this kind will reveal the bioprospecting potential of these Red Sea organisms as important sources of antifouling compounds.
The soft coral
Loopfuls from the agar slants were transferred to Zobell marine broth (ZMB; Himedia, India) in 10-ml tubes and incubated in a shaker incubator at 30°C overnight. Aliquots of the overnight cultures were transferred to conical flasks containing ZMB and were incubated at 30°C for 3 days in an incubator shaker. The cultures were centrifuged for 15 min at 5000 rpm. The supernatant was collected in a tube and filtered through a filter paper (Whatman no. 1) to remove the cells. An equal volume of ethyl acetate was added to the cell-free supernatant collected and agitated for 4 h at 28°C (Rajan & Kannabiran 2014). A separating funnel was used to separate the mixture and the organic phase was evaporated under reduced pressure using a vacuum evaporator to get the culture supernatant extract (CSE). The crude CSE was used for antibiofilm and antisettlement assays as well as GC-MS analysis.
A growth inhibition assay of CSE from the two bacterial isolates was performed against two biofilm-forming bacterial strains:
Final OD is the final optical density at 600 nm after 5 h of incubation, while initial OD is the initial optical density at 600 nm at baseline.
The biofilm inhibitory activity of the CSE was tested against the two target bacteria, PI is the percentage of biofilm inhibition, PF is the percentage of biofilm formation, BFC is the result of biofilm formed in the presence of the extract and C is the biofilm formed by the pure strain without the addition of extract.
The adult barnacles (
A toxicity assay was conducted in order to study the effects that the secondary metabolites present in the CSE had on the survival of the barnacle larvae. Six-well plates were used for the toxicity assay. Each well was filled with 5 ml of FSW and 25 nauplii (stage III) were added to the wells using a Pasteur pipette. Three different concentrations (5, 10 and 20 μg ml−1) of CSE were added to the treatment wells. The control wells were maintained without the addition of CSE. The 6-well plates were kept at 28°C in an environmentally controlled chamber (walk-in type). The number of dead/alive nauplii in the wells was observed periodically for 96 hours. The toxicity assay was repeated in triplicate (n=3 for each concentration) and the mean ± SD values were recorded.
An antisettlement assay was conducted to understand the settlement-inhibiting activity of the CSE against the settlement of barnacle larvae. The nauplii larvae were reared to the cypris stage in the laboratory. This experiment was also conducted in 6-well plates, with 25 cypris larvae in each well. The CSE (5 μg ml−1) was added to the treatment wells. The control wells were maintained without the addition of CSE. The 6-well plates were kept at 28°C in an environmentally controlled chamber (walk-in type) under dark conditions. The plate was observed periodically under a stereomicroscope for the settlement of larvae. The experiment was conducted in triplicate using different batches of barnacle larvae for a period of 48 h.
The bacterium isolated from the soft coral was labelled as MBR1, while the one from macroalga was labelled as MBR4. They were identified based on 16S rRNA gene sequencing. Previously outlined methods (Satheesh et al. 2012; Balqadi et al. 2018) were followed for the isolation of DNA and sequencing of the 16S rRNA. In brief, a loopful of bacterial culture was inoculated in marine broth in a conical flask and kept in an incubator at 30°C. The overnight bacterial culture was used for DNA extraction using a genomic DNA isolation kit (InstaGeneTM). The isolated DNA from the bacterial strains was confirmed using agarose gel electrophoresis. Universal 16S rRNA gene primers (27F:5′ - AGAGTTTGATCMTGGCTCAG - 3′ and 1100R: 5′ - GGGTTGCGCTCGTTG - 3′) were used for the amplification of the isolated DNA in a multigene thermal cycler (Labnet Inc). The PCR conditions were as follows: initial cycle at 95°C for 5 min, followed by 28 cycles at 95°C for 45 sec, annealing at 58°C for 45 sec, with extension at 72°C for 1 min 45 sec and a final extension step at 72°C for 10 min. The amplified PCR products were purified using a GeneJET PCR Purification Kit according to the manufacturer's instructions and then confirmed by agarose gel electrophoresis. The amplified PCR products were sent to Macrogen Inc. in South Korea for sequencing of the 16S rRNA gene. The resulting sequences were analysed and identified using the RDP classifier (
The secondary metabolites present in the CSE were analysed using gas chromatography-mass spectrophotometry (GCMS-QP2010 Plus, Shimadzu, Japan). The protocol outlined by Arulazhagan and Vasudevan (2009) was followed for the analysis. In brief, the capillary column (30 m × 0.25 mm; ID = 0.25 μm) was programmed as follows: the initial column temperature was set at 100°C for 1 min, then increased up to 160°C at the rate of 15°C min−1. The column temperature was then set for 7 min at 300°C, the final temperature, at a rate of 5°C min−1. Subsequently, with helium as the carrier gas at 1.0 ml min−1, the GC injector was held isothermally at 280°C flow rate while the temperature at the GC/MS interface was maintained at 280°C for an uninterrupted period of 3 min. The mass spectrometry was set to operate in electron impact ionization mode with 70 eV electron energy. The possible metabolites were identified with the National Institute of Standards and Technology library using the similarity index (<92%) by comparing their retention index.
The inhibitory activity against bacterial growth of different concentrations of CSE was tested using the Kruskal–Wallis H test. The antibiofilm assay results and the anti-larval settlement data were tested using one-way analysis of variance (ANOVA) in order to determine the difference between the controls and the treatments. Furthermore, the barnacle larval toxicity of CSE was tested using two-way ANOVA. Concentration and bacterial strain were used as the factors for two-way ANOVA. A post hoc Tukey HSD test was carried out to determine the variation between the controls and the treatment groups in the antibiofilm and antisettlement assays. All the statistical tests were conducted in STATISTICA (ver. 13) and
After the bacteria were screened using the agar overlay assay, one bacterium each was selected from soft coral and macroalgae. Based on its morphologic features and a comparison with the closest strain from the NCBI database, MBR1 had close similarity with more than 15 strains of
Phylogenetic tree showing the two organisms with other related species. The organisms from this study are highlighted in blue, while the red symbol was used to show the bootstrap values.
The CSE from the two bacterial strains inhibited the planktonic growth of the biofilm-forming bacteria
Bacterial growth inhibitory activity of the CSE of MBR1
Bacterial growth inhibitory activity of the CSE of MBR4
The percentage of biofilm inhibition from the extract against the test strains ranged from 45% to 78%, as presented in Figure 4. Both the lowest and high activity was recorded by the extract from MBR1, and the two different extracts demonstrated higher inhibition activity towards
Percentage of biofilm inhibition activity of the extracts from MBR1 and MBR4 against the biofilm bacteria
Post hoc Tukey test results (approximate probabilities) for the antifouling activity of CSE extracted from three bacterial strains against biofilm-forming bacteria and barnacle larvae
Bacterial extract | Antibiofilm activity | Antilarval selement activity | ||
---|---|---|---|---|
Cyprid larvae | ||||
Control | MBR1 | <0.001 | <0.01 | <0.001 |
MBR4 | <0.001 | <0.001 | <0.001 | |
MBR1 | MBR4 | 0.067 | 0.808 | 0.942 |
The CSE treatment did not show much effect on the survival of the nauplius larvae during the 96-h toxicity study (Figure 5). The two-way ANOVA results also confirmed the weak toxicity of the extracts against barnacle larvae, as evidenced by the insignificant variation (F=2.32; df=3,24;
Toxicity of CSE extracted from the bacterial strains against barnacle larvae
The results indicated reduced settlement of cypris larvae on the 6-well plates due to CSE treatment. The CSE of strain MBR4 exhibited higher settlement inhibition than the extract of MBR1 (Figure 6). The one-way ANOVA results indicated a significant variation in cyprid settlement between the control and CSE treatment groups (F=25.86; df=3,8;
Antilarval settlement activity of CSE against cyprid-stage larvae of barnacles
The GC-MS spectra of the CSE extracted from the two bacterial strains are presented in Figures 7 and 8. The secondary metabolites present in the CSE of MBR1 included n-tridecanol, 3-chloropropionic acid heptadecyl ester, hexadecanoic acid ester, hexacontane and tetrapentacontane (Table 2). The GC-MS analysis results of the CSE obtained from MBR4 showed the presence of compounds such as cyclopropane, octadecanoic acid ester, cis-anti-cistricyclo (2,6) dodecane −7,8-diol, 7-hexyltridecanol, tetrapentacontane, pentatriacontane, 2H-Pyran-2-one and 5-ethylidenetetrahydro-4-(2-hydroxyethyl) (Table 3).
GC-MS spectrum of CSE extracted from the bacterial strain MBR1
GC-MS spectrum of CSE extracted from the bacterial strain MBR4
Compounds detected in the CSE of MBR1
Compound name | RT (min) | Peak area |
---|---|---|
Hexane, 2,2-dimethyl- | 2.043 | 5682084 |
n-Tridecan-1-ol | 7.046 | 5873147 |
3-Chloropropionic acid, heptadecyl ester | 8.176 | 2029795 |
Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester | 8.627 | 13557111 |
Hexacontane | 8.808 | 5252629 |
Tetrapentacontane, 1,54-dibromo- | 9.027 | 2603872 |
Tetrapentacontane, 1,54-dibromo- | 9.2 | 4136789 |
Tetrapentacontane | 9.412 | 1932874 |
Tetrapentacontane | 9.832 | 13033013 |
Compounds detected in the CSE of MBR4
Compound name | RT (min) | Peak area |
---|---|---|
Pentane, 2,2,4-trimethyl- | 2.04 | 4716273 |
Cyclopropane, 1-(1-hydroxy-1-heptyl)-2-methylene-3-pentyl- | 8.542 | 3254671 |
Octadecanoic acid, 2,3-dihydroxypropyl ester | 8.633 | 5949198 |
(7R,8R)-cis-anti-cis-Tricyclo[7.3.0.0(2,6)]dodecane-7,8-diol | 8.783 | 6609750 |
Tetrapentacontane, 1,54-dibromo- | 8.945 | 7398782 |
7-Hexyltridecan-1-ol | 9.258 | 3273783 |
9,12,15-Octadecatrienoic acid, 2-phenyl-1,3-dioxan-2-yl ester, (Z,Z,Z)- | 9.35 | 4608587 |
Tetrapentacontane, 1,54-dibromo- | 9.426 | 4100361 |
17-Pentatriacontene | 9.549 | 3437915 |
2H-Pyran-2-one, 5-ethylidenetetrahydro-4-(2-hydroxyethyl)- | 9.777 | 3956877 |
The aim of this study was to investigate the antifouling activity of extracts of culture supernatant of surface-associated bacteria from soft coral and macroalgae collected from the Red Sea. The CSE from the isolated organisms showed antifouling activity in the form of potential antibiofilm and antisettlement activities against biofilm bacteria and barnacle larvae, respectively. Many previous studies have found that bacteria living on the surface of marine organisms such as sponges, corals and seaweeds produce compounds that protect the host from fouling organisms (Satheesh et al. 2016). The bacteria isolated from the surface of the marine organisms identified as
The CSE of the two isolates inhibited the growth of biofilm bacteria in liquid conditions based on turbidity measured with a spectrophotometer. This method is commonly used for determining bacterial growth under various conditions. For instance, Viju et al. (2014) and Ba-akdah & Satheesh (2021) reported the antibacterial activity of compounds obtained from bacteria associated with seaweeds using a similar method. Furthermore, this approach is relevant in easily determining whether a bioactive compound has antibacterial activity (Satheesh et al. 2012). It has been shown that the effect on antibiofilm formation is directly related to the compounds produced by the bacteria (Viju et al. 2014). The CSE tested in this study showed variation in the inhibitory effect against biofilm-forming bacteria and the tested organisms had varying susceptibility to the extracts. For example, the CSE from strain
After the growth of the biofilm bacteria, they adhere to the surface and colonise it to form biofilm. Bacteria are the initial colonisers on any newly exposed surface in marine waters, and they have been shown to influence the settlement of larval invertebrates (Hadfield 2010; Antunes et al. 2019; Dobretsov & Rittschof 2020; Peng et al. 2020; Chang et al. 2021). As a result, the adhesion assay utilising bacteria isolated from biofilms could be useful in developing natural products into antifouling agents. This makes targeting biofilm formation an important strategy for antifouling. The results of the antibiofilm assays conducted in this study indicated biofilm inhibitory activity of the compounds present in the CSE from the two bacterial strains. Both extracts demonstrated good antibiofilm formation activity. Natural antibiofilm compounds are required to tackle biofilm and its devastating effects in order to prevent the resulting substantial economic losses (Cámara et al. 2022).
Under laboratory conditions, the culture supernatant of bacterial strains isolated from a soft coral and a macroalga strongly inhibited the settlement of barnacle larvae without significant toxicity. Preventing settlement of larvae is an important requirement in the development of antifouling agents. The invertebrate larvae are responsible for the visible fouling structure seen on the surface of ship hulls (Vinagre et al. 2020). The mobile nauplius larva undergoes a series of transitions through stages before metamorphosising to the cypris stage, which then settles and attaches to suitable surfaces and finally transits to the sessile adult stage, contributing to biofouling (Kamino, 2016). The results of the study indicate that these extracts have an inhibitory effect on the settlement of the cyprid larvae, preventing metamorphosis to the adult stage and thereby preventing biofouling.
The activities demonstrated by the extracts are attributed to the secondary metabolites produced by the bacteria, as revealed by the GC-MS analysis. The GC-MS analysis of the CSE yielded many compounds with known bioactivities. Compounds detected from the
In conclusion, this study adds to our understanding of the ability of bacteria associated with the surface of marine organisms to produce secondary metabolites with antifouling activities. The diverse nature of the compounds presents in the CSE revealed the ecological functions of microbial association with marine organisms. Furthermore, the results indicate that the culture supernatant can be considered for the extraction of bioactive compounds. Further studies using other fouling organisms and field assays will provide more insights into the antifouling properties of the metabolites present in the culture supernatant.