Algicidal Activity and Stability of a Potential Biological Control Agent Against Harmful Algal Blooms

2.1. Algal cultures

Alexandrium catenella, Chattonella marina, Cochlodinium polykrikoides, and Heterosigma akashiwowere supplied by the National Fisheries Research & Development Institute (NFRDI), Republic of Korea; Akashiwo sanguinea, Fibriocapsa japonica, Gymnodinium impudicum, Isochrysis galbana, Prorocentrum micans, Prorocentrum minimum, and Scrippsiella trochoideawere obtained from the South Sea Institute of the Korea Ocean Research & Development Institute (KORDI), Republic of Korea; and Chlorella ellipsoidea (C-020), Chlorella vulgaris (C-012), Navicula elegans (B-225), Nannochloris oculata (C-031), Nitzschia pungens (B-037), Oscillatoria angustissima (CY-003), Pavlova gyrans (H-012), Phaeodactylum tricornutum (B-255), Skeletonema costatum (B-687), and Tetraselmis suecica (P-009) were provided by the Korea Marine Microalgae Culture Center, Busan, South Korea.

The dinoflagellate strains (A. sanguinea, A. catenella, C. polykrikoides, F. japonica, G. impudicum, P. micans, P. minimum, and S. trochoidea) and raphidophyte strains (C. marina and H. akashiwo) were maintained in f/2-Si medium (Guillard and Ryther, 1962) at 20°C and pH 8 under a 12 h dark/12 h cool white fluorescent light cycle (120 μmol photons m^–2s^–1). Strains classified as bacillariophyceae (N. elegans, N. pungens, P. tricornutum, and S. costatum) as well as live food organisms (C. ellipsoidea, C. vulgaris, I. galbana, N. oculata, O. angustissima, P. gyrans, and T. suecica) were maintained in the same f/2-Si medium at 20°C and pH 8, but under a lower light intensity (30 μmol photons m^–2s^–1).

2.2. Isolation and screening of algicidal bacteria

Water samples were collected from coastal surface waters in Jinhae Bay, South Korea. Samples were serially diluted with sterile seawater, and 0.1 mL aliquots of each dilution were spread onto PPES-II agar plates (Taga, 1968). After incubation at 20°C for seven days, individual colonies exhibiting distinct morphologies were re-streaked onto fresh PPES-II agar plates for purification and subsequently stored at –70°C in 20% glycerol.

The isolated bacteria were precultured in 10 mL of PPES-II broth at 20°C with shaking at 200 rpm for 48 h. For screening purposes, C. marina (1.0 × 10⁴ cells/mL, mid-exponential phase) was used. In each well of a 24-well plate, 1 mL of algal culture was combined with 0.2 mL of bacterial culture (or filtrate or medium control), and the plates were monitored microscopically at ×200 magnification. A bacterial strain exhibiting marked algicidal activity against C. marina was selected for further study.

2.3. Identification of bacterium by phylogenetic analysis

Morphological observations were performed via Gram staining, and conventional biochemical tests were conducted according to the methods described by Smibert and Krieg(1994). Bacterial strain was identified by PCR amplification of the 16S rRNA gene, followed by BLAST analysis against sequences in the GenBank database.

Two oligonucleotide primers, as described by Dunbar et al.(2000), were employed: (Forward) 5'-AGAGTTTGATCCTGGCTCAG-3' and (Reverse) 5'-TACCTTGTTACGACTT-3'. PCR was conducted using intact cells that were heat-treated at 95°C for 5 min as the template. The thermal cycling conditions consisted of 25 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C), and extension (2 min at 72°C), with a final extension at 72°C for 5 min. The resulting 16S rDNA amplicon was purified, cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), and sequenced. Sequence comparisons were performed using the BLASTN tool (http://www. ncbi.nlm.nih.gov/BLAST) at the National Center for Biotechnology Information (NCBI). Alignments were generated using CLUSTAL W, and a phylogenetic tree was constructed with MEGA version 7.0 (Kumar et al., 2016).

2.4. Relationship between bacterial growth and algicidal activity

The growth curve and algicidal activity of the DC-25 strain were monitored at 3 h intervals over a 36 h period. The DC-25 strain was inoculated into PPES-II broth (initial cell concentration: 10³ cells/mL) and incubated under optimal conditions (25°C, pH 7.0, 2.0% NaCl) with shaking at 200 rpm; optical density (OD) measurements were taken every 3 h. The algicidal activity of the culture filtrate, as well as that of bacterial cells from each growth phase, was evaluated by adding the filtrate to C. marina cultures (1.0 × 10⁴ cells/mL) in a 24-well plate under the same algal culture conditions.

The bioassay plates were incubated under the previously described algal culture conditions, and the number of viable, motile cells in each well was counted using a Sedgewick-Rafter chamber and an inverted microscope. The algicidal activity of DC-25 strain was calculated, using the following equation (Kim et al., 2007): Algicidal activity (%) = (1–Tt/Ct) × 100. Tt (treatment) and Ct (control) are the cell concentrations of C. marina with culture filtrate of DC-25 strain, and sterile PPES‑II broth, respectively, after inoculation time (t).

2.5. Algicidal spectrum of culture filtrate from bacterial DC-25 strain

The culture filtrate of the DC-25 strain was prepared as described above. Its algicidal spectrum was evaluated against a range of harmful algal bloom (HAB) species and live food organisms, including A. sanguinea, A. catenella, C. marina, C. ellipsoidea, C. vulgaris, C. polykrikoides, F. japonica, G. impudicum, H. akashiwo, I. galbana, N. oculata, N. elegans, N. pungens, O. angustissima, P. gyrans, P. tricornutum, P. micans, P. minimum, S. trochoidea, S. costatum, and T. suecica.

For each species at the mid-exponential growth phase (1.0 × 10⁴ cells/mL), 5% (v/v) of the culture filtrate was added. The test flasks were then incubated under the previously described algal culture conditions for 12 h. Algicidal activity was quantified using the established equation.

2.6. Thermal and pH stability of the algicidal activity

The thermal and pH stability of the algicidal activity of Arthrobacter sp. DC-25 were evaluated under various conditions. For thermal stability assessment, culture filtrates from bacterial cultures grown in PPES-II broth at 30°C were incubated at 4, 20, 40, 50, 70, and 100°C for 30 minutes. For pH stability, the filtrates were adjusted using 0.1 M citrate-phosphate buffer (pH 3–7) or 0.1 M Tris-HCl buffer (pH 8–10) and incubated for 30 minutes. Following these treatments, 5% (v/v) of the filtrates was added to C. marina cultures (1.0 × 10⁴ cells/mL) to determine the residual algicidal activity. The relative algicidal activity was expressed as a percentage, with activity at 4°C and pH 7.0 or 8.0 set as 100%. PPES-II broth subjected to the same treatments was used as a control. Data are presented as the mean ± standard deviation from triplicate assays.

3.1. Screening of algicidal bacteria

A total of 42 bacterial strains were isolated, of which four exhibited algicidal activity against C. marina. The algicidal activity of these four strains was evaluated, and strain DC-25 demonstrated the highest efficacy, reducing C. marina cell density by 98%.

3.2. Identification and characterization of DC-25 strain

To genetically characterize strain DC-25, the 16S rRNA gene was amplified and sequenced, followed by alignment with available sequences in the GenBank database. Sequence analysis revealed that strain DC-25 exhibited high similarity to both Arthrobacter oxydans DSM 20119ᵀ (X83408) and A. polychromogenes DSM 20136ᵀ (X80741), with nearly identical sequence identity (99.2%). Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain DC-25 belongs to the genus Arthrobacter (Fig. 1). The neighbor-joining tree showed that DC-25 formed a distinct clade, closely related to A. polychromogenes DSM 20136ᵀ with a bootstrap support value of 98%. The strain was positioned within a well-supported cluster that included A. oxydans, A. ureafaciens, and A. ilicis, further supporting its taxonomic placement within the genus.

Fig. 1.

Phylogenetic tree based on 16S rRNA gene sequences of the isolated DC-25 strain and closely related species in the genus Arthrobacter. The numbers at the nodes indicate bootstrap support values (%), derived from neighbor-joining analysis of 1,000 resampled datasets. The scale bar represents 0.005 nucleotide substitutions per site.

Physiological characterization revealed that Arthrobacter sp. DC-25 exhibited optimal growth at 25°C, pH 7.0, and 2.0% salinity (Fig. 2). The strain did not grow below 10°C or at pH values lower than 5.0 but demonstrated tolerance to a range of salinity conditions.

Fig. 2.

Effects of temperature, initial pH, and NaCl concentrations on the growth of Arthrobacter sp. DC-25 in PPES-II medium.

Although numerous algicidal bacteria have been isolated from various environments, they are predominantly affiliated with the Cytophaga-Flavobacterium-Bacteroidetes (CFB) group or γ-Proteobacteria (Nakashima et al., 2006) or the genus Pseudoalteromonas (Mu et al., 2007a). In contrast, Arthrobacter species, which are Gram-positive bacteria, have been rarely reported to exhibit algicidal activity in marine environments. Previous studies have demonstrated that other Gram-positive genera, such as the genus of Bacillus, can produce algicidal metabolites effective against harmful algae (Mu et al., 2007b; Jeong and Son, 2021). Given that Gram-positive bacteria are typically found in deep-sea sediments rather than in surface waters (Li et al., 1999), their role in algicidal interactions may have ecological significance, potentially functioning as a top-down control mechanism in marine ecosystems (Liu et al., 2008).

The identification of Arthrobacter sp. DC-25 as an algicidal strain expands the understanding of Gram-positive bacteria in algal bloom control. Its genetic and physiological characteristics suggest that it may play a distinct role in regulating harmful algal blooms (HABs), warranting further investigation into its algicidal mechanisms and potential environmental applications.

3.3. Relationship between bacterial growth and algicidal activity of DC-25 strain

Fig. 3 illustrates the growth curve of Arthrobacter sp. DC-25 under optimal culture conditions (25°C, pH 7.0, 2.0% NaCl) and the corresponding algicidal activity of the culture filtrate at different bacterial growth phases against C. marina. The bacterial growth curve (represented by optical density at 640 nm) exhibited a typical sigmoidal trend, with an initial lag phase observed from 0 to 6 h. This was followed by an exponential growth phase from approximately 6 to 18 h, where a sharp increase in bacterial density was detected. The growth rate then gradually slowed, and the stationary phase was reached after 24 h, maintaining a high cell density until the end of the observation period (36 h).

Fig. 3.

Growth curve of Arthrobacter sp. DC-25 at optimal culture conditions (25°C, pH 7.0, 2.0% NaCl) and algicidal activity by the culture filtrate of each growth phase of Arthrobacter sp. DC-25 against Chattonella marina. -■- = bacterial growth curve (O.D. at 640 nm). Bar graph= algicidal activity (control = algal cultures with PPES-II broth added).

The algicidal activity of the culture filtrate displayed a time-dependent pattern, increasing progressively with bacterial growth. Notably, a significant algicidal effect was first observed at 9 h, coinciding with the early exponential phase of bacterial growth. The algicidal activity continued to increase, peaking at 24 h, which corresponded to the stationary growth phase. Beyond this point, the algicidal activity remained stable, with no substantial decline observed up to 36 h.

The results indicate that Arthrobacter sp. DC-25 exerts its algicidal effect through an indirect mechanism, releasing algicides into the surrounding environment. Indirect algicidal mechanisms, observed in approximately 70% of algicidal bacteria (Mayali and Azam, 2004; Roth et al., 2008), provide a competitive advantage by allowing bacteria to target a broad range of harmful algal species without direct interaction. Consistent with previous studies demonstrating a correlation between bacterial cell density and algicidal activity (Nakashima et al., 2006; Mu et al., 2007b), Arthrobacter sp. DC-25 exhibited its highest algicidal activity during the late exponential and early stationary phases. This suggests that the algicidal compounds produced by this strain are likely secondary metabolites synthesized in the later stages of growth. The sustained high algicidal effect during the stationary phase further implies that these compounds are either continuously produced or remain stable in the culture filtrate over time. Further research is needed to elucidate their chemical nature and stability.

3.4. Algicidal spectrum of culture filtrate from DC-25 against HAB species

The algicidal spectrum of culture filtrate from Arthrobacter sp. DC-25 against various harmful algal bloom (HAB)-forming species was evaluated, and the results are presented in Fig. 4. The data indicate that the algicidal activity of Arthrobacter sp. DC-25 varies significantly depending on the target algal species.

Fig. 4.

Algicidal spectrum of Arthrobacter sp. DC-25 against various HAB-forming species. The algicidal activity of Arthrobacter sp. DC-25 was assessed against multiple harmful algal bloom (HAB)-forming species. A 5% culture filtrate was introduced into each algal culture at the mid-exponential growth phase, and algicidal activity was evaluated after 12 h. The control group consisted of algal cultures supplemented with sterile PPES-II broth. Data are presented as mean ± standard deviation, based on triplicate assays.

Among the tested species, A. catenella and C. marina exhibited the highest susceptibility to Arthrobacter sp. DC-25, with algicidal activity exceeding 98%. This suggests that Arthrobacter sp. DC-25 produces bioactive compounds or induces physiological stress that effectively suppresses these algal species. Similarly, A. sanguinea and G. impudicum showed moderate levels of algicidal activity, ranging from 50% to 70%, indicating a potential species-specific mode of action. In contrast, F. japonica, H. akashiwo, and S. trochoidea exhibited relatively low susceptibility to Arthrobacter sp. DC-25, with algicidal activity below 20%. The Prorocentrum species (P. micans and P. minimum) and C. polykrikoides also demonstrated minimal algicidal responses, suggesting either resistance to the bacterial metabolites or limited interaction between Arthrobacter sp. DC-25 and these algal species.

Table 1.

Algicidal effects of Arthrobacter sp. DC-25 on the growth of Bacillariophyceae and live food organisms

The differential susceptibility among HAB species may be attributed to variations in cell wall composition, physiological defense mechanisms, or differential responses to bacterial-secreted compounds. Previous studies have shown that algicidal bacteria can exhibit species-specific activity through the production of bioactive compounds. For example, Umetsu et al.(2019) reported that Alteromonas sp. produced the algicidal compound Questiomycin A, which effectively inhibited the growth of C. antiqua. Similarly, the strong algicidal effect of Arthrobacter sp. DC-25 against A. catenella and C. marina suggests the presence of potent bioactive metabolites targeting these HAB species.

These findings highlight the potential of Arthrobacter sp. DC-25 as a candidate for HAB mitigation strategies. Future investigations should focus on identifying the specific algicidal compounds produced by Arthrobacter sp. DC-25 and elucidating the molecular mechanisms underlying its selective activity. Furthermore, additional studies should assess its effectiveness under natural environmental conditions and evaluate any potential ecological risks associated with its application in marine ecosystems.

3.5. Algicidal effects of culture filtrate from DC-25 on the growth of Bacillariophyceae and live food organisms

The algicidal effects of the culture filtrate from Arthrobacter sp. DC-25 were assessed on various algal species, including Bacillariophyceae, Chlorophyceae, Cyanophyceae, Haptophyceae, and Prasinophyceae. The results indicated that Arthrobacter sp. DC-25 did not exhibit algicidal activity against any of the tested species, as all results were negative. Notably, among Bacillariophyceae, N. pungens, a known harmful algal bloom (HAB)-forming species, showed an increase in cell density, suggesting a potential stimulatory effect rather than inhibition, while other HAB-associated species, such as S. costatum, remained unaffected. Similarly, no algicidal effects were observed in Chlorophyceae (C. ellipsoidea, C. vulgaris, and N. oculata), Cyanophyceae (O. angustissima), Haptophyceae (I. galbana and P. gyrans), or Prasinophyceae (T. suecica).

Although indirectly acting algicidal bacteria often exhibit species-specific effects mediated by chemical compounds (Yoshinaga et al., 1997; Jeong and Son, 2021), Arthrobacter sp. DC-25 did not display strict species specificity but instead demonstrated a broad host range. While no algicidal effects were observed on Bacillariophyceae and live food organisms, the strain exhibited strong algicidal activity against A. catenella and C. marina, with no adverse effects on beneficial marine species. The mechanism underlying this selective activity may be associated with bacterial secondary metabolites that disrupt algal cell integrity. For example, mycosubtilins produced by bacteria in the genus Bacillus interact with the cytoplasmic membrane, resulting in increased ion permeability and lysis of both dinoflagellate and raphidophyte species (Jeong and Son, 2021). Similarly, the strong activity of Arthrobacter sp. DC-25 against A. catenella and C. marina suggests that it may produce bioactive compounds with a mode of action distinct from those affecting Bacillariophyceae.

This selective algicidal activity highlights the potential of Arthrobacter sp. DC-25 as a targeted biological control agent for HABs. Further investigations are needed to determine whether different environmental conditions or bacterial metabolites could influence its algicidal potential, as well as to identify the specific bioactive compounds responsible for its selective effects.

3.6. Thermal and pH stability of the algicidal activity of Arthrobacter sp. DC-25

The thermal and pH stability of the algicidal compounds produced by Arthrobacter sp. DC-25 were evaluated under different conditions (Table 2). For thermal stability, the culture filtrate was incubated at 4, 20, 40, 50, 70, and 100°C for 30 minutes before activity measurement. For pH stability, the filtrate was adjusted to pH 3–10 using appropriate buffers and incubated for 30 minutes prior to assessment.

Table 2.

Thermal and pH stability of the algicidal activity of Arthrobacter sp. DC-25

The results indicate that the algicidal activity of Arthrobacter sp. DC-25 remained relatively stable at moderate temperatures (20–50°C), with only a slight reduction observed at elevated temperatures (70°C: 87.0 ± 7.4%; 100°C: 78.0 ± 9.2%). In terms of pH stability, the algicidal activity remained high in neutral to slightly alkaline conditions (pH 6–9) but declined significantly under more acidic conditions (pH 3–5), with the lowest activity recorded at pH 3.0 (34.6 ± 11.6%).

These findings suggest that Arthrobacter sp. DC-25 exhibits strong thermal stability within a moderate temperature range and maintains optimal algicidal activity under neutral to slightly alkaline conditions. Given that the average pH and temperature of seawater are approximately 8 and 5°C, respectively, the pH stability and heat tolerance of Arthrobacter sp. DC-25 may provide an ecological advantage in harmful algal bloom (HAB) management.

Unlike chemical control methods, which can negatively impact non-target organisms and disrupt ecosystems, algicidal bacteria offer a more sustainable and environmentally friendly alternative. Arthrobacter sp. DC-25, in particular, demonstrates strong algicidal activity while exhibiting minimal toxicity toward beneficial marine species. These characteristics highlight its potential as a biological control agent for mitigating HABs.

Further research is necessary to identify and characterize the specific algicidal compounds responsible for the observed effects. Understanding their molecular mechanisms, stability, and mode of action will facilitate the development of optimized application strategies for HAB mitigation. Additionally, large-scale field studies are required to assess the efficacy and ecological impact of introducing Arthrobacter sp. DC-25 into natural marine environments. With continued investigation, algicidal bacteria such as Arthrobacter sp. DC-25 could become a key component of integrated HAB management strategies, offering an effective and ecologically sustainable solution to harmful algal blooms.