Physico-chemical factors of Belawan mangrove forest waters
The physicochemical factors of the water were measured at each sampling location. The physicochemical factors were relatively similar among the study sites, as shown in Table 1.

Table (1):
Physicochemical data of Belawan mangrove waters at the three sampling stations

TDS, total dissolved solids; DO, dissolved oxygen; BOD, biological oxygen demand; COD, chemical oxygen demand

The pH of the water at the sampling locations at stations I and II was 6.8, while that at station III was 7.0. According to Mustapha and Halimoon,¹⁹ the optimum pH for bacterial growth is 6.5-7.5, which indicates that the sampling location was the correct pH for obtaining neutrophilic bacterial isolates. The optimum pH for the hydrocarbon biodegradation process ranges between 6.0-8.0, which indicates that the bacteria in the Belawan mangrove waters are environmentally adapted to support the hydrocarbon degradation process. The water temperature at the three stations was 28.5, 29.8, and 30.0 °C, which is normal. At this temperature, the thriving bacteria were mesophilic. The highest salinity was observed at station I (1.5%), which was likely due to the closer proximity to the high seas than the other sites. The highest turbidity was at station III (2 NTU), with a TDS value of 8.14 mg/L, which could be attributable to the close proximity to settlements (contaminated by household waste).

According to Ikhtiar et al.²⁰ the higher the DO value, the better the water quality, whereas the higher the BOD value, the lower the water quality. Based on the regulations of the State Minister of Environment No. 03 of 2010, the maximum allowable levels of BOD and COD is 50 mg/L and 100 mg/L, respectively. The highest DO, BOD, and COD values were at Station III (8.17 mg/L), Station I (0.48 mg/L), and Station II (5.6 mg/L), respectively.

According to Patty et al.²¹ water P and N contents are crucial for organism growth and are indicators of water quality. If their thresholds are exceeded, it can be deadly to several types of marine life. The main sources of P and N in the water is plants or organism decomposition; however, it can also originate from industrial waste containing organic compounds. The highest P and N contents were at Station II (0.48 mg/L) and Station I (5.5 mg/L), respectively. The P and N levels at the three stations were high based on the Decree of the Minister of the Environment, which stipulates that the quality standard for P and N concentrations are 0.015 ml/L and 0.008 mg/L, respectively, indicating that the mangrove waters of Belawan are polluted.

Culturable bacterial isolates
Bacteria were isolated and purified using NA, a general medium for bacterial cultivation. A total of 29 different morphotype isolates were successfully purified from 3 sampling sites. Using Gram staining, macroscopic colony observations included color, shape, edge, elevation, surface, and consistency. The results showed that the number of purified isolates from each station was relatively similar: ten isolates from stations I and III and nine isolates from station II. The macro-and micro characteristics of the isolates are listed in Table 2.

Table (2):
Macro-and microscopic characteristics of bacteria isolated from the waters of the Belawan mangrove forest

(+) = Gram-positive; (-) = Gram-negative

The isolate colors were pale yellow (n = 1), white (n = 3), clear white (n = 3), yellowish white (n = 5), and milky white (n = 17). Based on the colony shape, 13 isolates were circular and 16 were irregular. The edges of the colonies on each isolate included three isolates with undulated edges and 27 isolates with entire edges. There were 3 isolates with elevated elevation, 6 umbonate, and 20 convex. Nineteen isolates had smooth surfaces and 10 isolates had rough surfaces. Three isolates exhibited a mucoid (slimy) consistency and the others were butyrous (not slimy/creamy). Fourteen isolates were Gram-negative and 15 were Gram-positive. Twenty-six isolates were bacillus-shaped cells and three coccobacilli.

Molecular identification
16S rRNA gene amplification using primers 27F and 1492R, visualized by electrophoresis, were observed on a 1% agarose gel using a 1 kb marker. All the bacterial isolates showed DNA bands measuring approximately 1500 bp in length (Figure 2).

The sequence results were then compared with data from GenBank in the National Center for Biotechnology Information (NCBI) database (Table 3).

Table (3):
Results of BLAST analysis of bacterial isolates from the Belawan mangrove waters.
The BLAST analysis conducted on bacteria isolated from the Belawan mangrove waters revealed the presence of 8 distinct genera, which collectively represent a total of 15 different bacterial species

Based on the results of the Basic Local Alignment Search Tool (BLAST) analysis of bacterial isolates from the waters of the Belawan mangrove forest, eight species were isolated from station I: Myroides profundi, Enterobacter kobei, Shigella sonnei, Shigella flexneri (two isolates), Bacillus anthracis, Pseudomonas hydrolytica, Providencia huaxiensis, and Bacillus cereus. At station II, five species were detected: Bacillus cereus, Aeromonas caviae (two isolates), Aeromonas salmonicida, Pseudomonas balearica, Bacillus cereus (four isolates), and Bacillus mycoides. Five species were isolated from station III, namely Bacillus cereus (six isolates), Klebsiella pneumoniae, Enterobacter cloacae, Aeromonas caviae, and Pseudomonas khazarica.

Various bacterial genera have been isolated from mangrove waters, as reported in previous studies, including Bacillus, Nitrococcus, Planococcus, Lactobacillus, Pseudomonas, Micrococcus, Vibrio, Listeria, and Paenibacillus.^22-25 These findings highlight the bacterial diversity in mangrove water ecosystems, which aligns with the results of our study. A diagram illustrating the bacterial species isolated from the Belawan mangrove forest at three sampling locations is presented in Figure 3.

Seven species were found only at station I, namely B. anthracis, E. kobei, M. profundi, P. huaxiensis, P. hydrolytica, S. flexneri, and S. sonnei. All the species found at Station I were bacteria found in clinical samples from humans or animals. This could be due to the location of Station I, which is drained by a small river that may carry microorganisms from the mainland. In addition to clinical samples, all species can tolerate the temperature and salinity conditions present at Station I.²⁶ Furthermore, Zhu et al.²⁷ reported that apart from clinical specimens, the Enterobacter group can grow at temperatures of 15-42 °C. The optimal temperature of S. sonnei is 37 °C, pH 5.0-6.0, and salinity 1-4%.²⁸ Mallick et al.²⁹ stated that the optimal temperature of S. flexneri is 37 °C. The optimal temperature for the growth of P. huaxiensis is 35-37 °C and pH 6.0-7.0.³⁰ P. hydrolytica grows optimally at 37 °C, pH 7.0, and has a salinity tolerance of more than 4%. The environmental conditions at Station I supported the growth of the bacterial species present there.

The species A. salmonicida, B. mycoides, and P. balearica were found at station II, which was surrounded by mangroves and received direct input from the river flow. These three species were able to live at station II, which was 29 °C and 1.3% salinity. P. balearica was first isolated on the Balearic Islands, which can grow at temperatures up to 45 °C and salinity up to 8.5%,³¹ which is supported by Nejad et al.³² who stated that P. balearica is a salt-tolerant bacterium. Station III was located close to the residential areas. Three isolates were found at Station III: K. pneumoniae, E. cloacae, and P. khazarica, which may have been influenced by the presence of waste generated by human and animal activities. Several researchers have reported that these three isolates can be isolated from several samples related to human and animal life. Klebsiella can be isolated from clinical samples (the urine and blood of patients with urinary tract infections and bacteremia), surface water,³³ and sediment and seawater samples.³⁴ E. cloacae can be found in diseased rice seedlings,³⁵ activated sludge from municipal wastewater treatment plants,³⁶ and sediment and seawater samples.³⁴ P. khazarica was first isolated from the Khazar Sea, and decomposes polycyclic aromatic hydrocarbons. This strain is able to grow without NaCl, tolerates up to 8.5% NaCl, and can grow at pH 3.0-10.0 (optimal pH 6.0-7.0) at a temperature of 10-45 °C (optimally 30 °C).

The species A. caviae was detected at stations II and III. Based on the physical and chemical factors at stations I and III, which were not significantly different, the adjacent location points allowed the presence of the same species at these two locations. Although it receives direct inputs from river flows carrying various compounds and wastes, this species can still grow. This is supported by Shamim et al.³⁷ who reported that A. caviae isolated from estuarine surfaces in India showed resistance to lead and tolerance to several heavy metals, including Zn, Cd, Cu, and Hg.

B. cereus was isolated from all three stations and dominated the water in the Belawan mangrove forest. This may be because B. cereus naturally thrives on many substrates. Many studies have isolated this species from various locations, one of which is Hemalatha and Banu³⁸ who isolated B. cereus from six different samples, including soil, wastewater, air, freshwater, seawater, and milk. Based on the results of this study, the waters of the Belawan mangrove forest were predominantly inhabited by Bacillus species. This finding is consistent with the research conducted by Alhelaify et al.,³⁹ who studied bacterial diversity in mangrove environments in Saudi Arabia and reported that Bacillus spp. accounted for up to 50% of the isolated bacteria. Bacillus spp. play a crucial role in the decomposition of mangrove litter, contributing to nutrient cycling and ecosystem sustainability.

The bacterial DNA sequences obtained from the Belawan mangrove waters were then used to create a phylogenetic tree to determine their level of kinship and genetic evolutionary distance using the MEGA X software (Figure 4). Based on the phylogenetic tree, the bacterial species from the waters of the Belawan mangrove forest had very high genetic variation. Each end of the phylogenetic tree represents a unique species in all isolates. Bacillus cereus, a species that dominates the waters of the Belawan mangrove forest, has very high genetic variation. Water P content is not directly related to bacteria but is related to the level of water fertility. Excessive nutrients in the ecosystem can be caused by high N and P contents from the decomposition of organic matter or human activities that produce waste, which can cause eutrophication. The high N and P contents at the three stations, due to exposure to toxic compounds and waste carried by the river, allowed this species to mutate and survive environmental changes. The high genetic variation and the results obtained were then tested for their potential to decolorize dye waste.

Decolorization potential
Screening potential for decolorization activity of isolates was performed in MSM with liquid dye waste as a carbon source using concentrations of 25-100%. Bacterial growth was measured based on the diameter of the colony after four days of incubation. Relative growth responses are shown in Table 4.

Table (4):
Colony growth diameter scale of bacterial isolates from Belawan mangrove waters on solid mineral salt medium (MSM) at different concentrations of dye waste (25%, 50%, 75%, and 100%)

All bacterial isolates grew on solid MSM with a concentration of 25% dye waste; however, not all isolates grew at concentrations of 50%, 75%, and 100%. Eleven isolates grew very well on media with 25% and 50% dye waste concentrations, eight isolates grew on media with 75% dye waste concentration, and only five isolates grew on media with up to 100% dye waste concentration.

Each isolate showed a different colony diameter growth scale, which could be due to the ability of each isolate to use the dye waste contained in the test medium as the only carbon source. However, an increase in the concentration of dye waste also affected colony growth. The higher the concentration of dye waste, the lower was the growth of bacterial cells in the test medium. This could be because the dye contained in the waste used is a synthetic compound that is toxic to bacteria; thus, bacterial isolates with a high colony diameter scale are considered better for using dye waste in their growth as the only carbon source. Therefore, isolates with the highest colony diameter scale and able to grow up to 100% concentration of dye waste were selected and tested for their decolorization ability on liquid MSM media, they were A4 (S. flexneri), A10 (B. cereus), B4 (A. salmonicida), C4 (K. pneumoniae), and C6 (E. cloacae).

Decolorization activity
The decolorization activity of the five potential isolates from the screening process was evaluated in liquid MSM with dye waste as the carbon source. Qualitatively, decolorization activity was observed by reducing the color intensity of the culture medium during cultivation. Qualitatively, the rate of decolorization of dye waste was evaluated by measuring the decrease in the absorbance of the culture media at 365 nm. The decolorization activity profile of the five isolates after cultivation for 15 days is shown in Figure 5.

Five species exhibited the ability to reduce absorbance values in media containing 50% dye waste. Decolorization ranged between 12-21%, and the color reduction profile was similar during cultivation among the isolates. No decrease of absorbance value was detected for control until 15 days. A. salmonicida and E. cloacae exhibited slightly better decolorization than the other three isolates. Even at low decolorization, compared with our previously reported cases for fungi, the results of this study clearly indicate that the bacteria used the carbon available in the liquid waste. The decolorization rate was determined by several factors, of which the concentration and toxicity of the dyes are among the most important. Different decolorization activities have been reported for bacteria and fungi using various dyes. For instance, Moosvi et al.⁴⁰ reported that the decolorization of azo dyes by bacteria with the addition of glucose showed a maximum decolorization of 94%, whereas without glucose it was only 40%, this could be due to glucose metabolism resulting in the production of reduced nucleotides (NADH and FADH) causing an increase in decolorization efficiency. Seyedi et al.⁴¹ also reported that in the decolorization of dyes by bacteria of the genus Halomonas, the highest percentage of decolorization was achieved with the addition of supplements (glucose, peptone, and yeast extract). The percentage decolorization by the strain requires a certain glucose concentration. Decolorization increased with an increase in glucose concentration up to 10%, followed by a decrease with an increase in glucose concentration. Low glucose concentrations (1-5%) did not support the growth of bacteria in the test medium, and at higher concentrations
(>10%), bacteria preferred to use glucose.

The bacterium A. salmonicida induces degradation and decolorization. A. salmonicida has five enzymes that have the potential to degrade chitin,⁴² which are resistant to lauric acid, dyes, and aromatic amines. Furthermore, these enzymes have shown technical feasibility for regenerating biomaterials during the decolorization of wastewater.⁴³ Wanyonyi et al.⁴⁴ reported that B. cereus, which produces proteases, has a decolorization efficiency of up to 98% within 24 hrs with a dye concentration of 1.0 × 10^-5 M. The lactase enzyme produced by K. pneumoniae also exhibited high decolorization and oxidation efficiencies for azo and malachite green dyes under acidic and neutral conditions.⁴⁵ Chantarasiri⁴⁶ isolated K. pneumoniae and Enterobacter sp. from mangroves and found that they showed strong decolorization efficiencies within 72 hrs of incubation with 0.01% reactive dye, with decolorization percentages of 20% and 92%, respectively. E. cloacae effectively decolorizes azo dyes and anthraquinones with different structures, the decolorization rate increases with increasing temperature from 20 °C to 30 °C .⁴⁷ The potential of the S. flexneri strain for the decolorization of dye waste has not yet been reported; however, Said et al.⁴⁸ reported that Shigella was able to use polycyclic aromatic hydrocarbons (PAHs) containing up to four rings as the sole carbon source.

Conclusively, this study isolated 29 bacterial species from the waters of the Belawan mangrove forest. Based on molecular identification, the isolates consisted of eight genera of bacteria from 15 different species: A. caviae (three isolates), A. salmonicida, B. anthracis, B. cereus (12 isolates), B. mycoides, E. cloacae, E. kobei, K. pneumonia. The bacterial species Profundi, P. huaxiensis, P. balearica, P. hydrolytica, P. khazarica, S. flexneri (two isolates), and S. sonnei. Based on the screening test for potential dye waste in solid MSM, five different bacterial species with the best growth potential were obtained: Shigella flexneri, Bacillus cereus, Aeromonas salmonicida, Klebsiella pneumoniae, and Enterobacter cloacae. The percentage of dye waste decolorized for 15 days with the addition of 1% glucose showed a better decolorization ability than that without glucose. The best decolorization ability was shown by Aeromonas salmonicida, with a percentage value of decolorization on two types of media: 36.2% with glucose and 25.0% without glucose.