A Research on the Diversity and Enzyme Productivity of the Salt-Resistant Microorganisms Isolated from the Nakdong River Estuary Wetland for the Search of Fermentation Strains

Screening of the halotolerant bacteria was done from the water samples of freshwater wetlands from Samrak Wetland Ecological Garden (35°10'25.8"N 128°58'21.9"E) and Daejeo Ecological Park (35°11'57.7"N 128°58'35.9"E), two representative wetlands of the Nakdong River Estuary in Korea. Isolation of halotolerant bacteria was done by serial diluting each sample in 0.85% saline buffer 10^-1 to 10^-4 and aerobically cultivating the plates at 37 °C using Marine agar medium (BD, New Jersey, USA). After cultivation, the same solid medium was used for further single colony isolation of halotolerant bacteria based on the morphological properties of the colonies. Pure colonies were obtained after several transfers, and the growth of the isolated bacteria at 37 °C on complex media were tested using Nutrient agar (BD, New Jersey, USA), R2A agar (BD, New Jersey, USA), and tryptic soy agar (BD, New Jersey, USA). As a halotolerant bacteria, the growth in the presence of 5-10% sodium chloride concentration was tested using marine agar medium, and optimal condition was confirmed. Also, optimum pH condition was confirmed using the same medium prepared with different pH at 5, 7, 9.

For bacterial identification, strains isolated from domestic wetland freshwater samples under aerobic conditions were cultivated on marine agar medium, and plates were sent to BIOFACT Co., Ltd. for 16S rDNA sequencing. Taxonomic identification based on the 16S rDNA sequence of the strains was analyzed using 16S-based ID service in EzBioCloud Database (https://www.ezbiocloud.net/) developed by Chun- Lab Inc. Phylogenetic analysis was done using BioEdit and MEGA X program.

Hydrolytic enzyme (amylase, lipase, protease) activities of the isolated halotolerant bacteria were tested on a selective agar medium supplemented with specific substrates to react accordingly. Hydrolytic enzyme activity was identified by cultivating the spot inoculated plates with halotolerant isolates using marine agar medium supplemented with 0.2% soluble starch (BD, New Jersey, USA), 1% Tween 80 (Sigma-Aldrich, Massachusetts, USA), and 2% skim milk (BD, New Jersey, USA) for amylase, lipase, protease activity, respectively. After 7 days of incubation at 37 °C, the plates were examined for enzyme activities by size of the clear zone around colonies. The area of the clear zone was determined as total area minus colony area (+++: > 7 mm, ++: 4-6 mm, +: 1-3 mm).

The auxin activity assay was performed using Salkowski reagent after incubating the isolates at 37 °C for 5 days in marine broth (BD, New Jersey, USA) supplemented with 0.1% L-tryptophan (Sigma-Aldrich, Massachusetts, USA). 800 μl of Salkowski reagent (35% HClO₄, 2% FeCl3) was added to 400 μl culture supernatant and incubated in the dark for 30 mins. Auxin activity was measured by the change of the color (Red: ++; Pale red: +; Orange: w; Colorless: -).

Halotolerant bacteria with possible industrial capability for food and cosmeceutical market was isolated from domestic Nakdong River estuary wetland freshwater samples (Samrak Wetland Ecological Garden and Daejeo Ecological Park wetland freshwater) aerobically culturing the samples using marine agar medium. Pure single colonies were obtained by streak plate method based on their morphological chracteristics. Screening result, as shown in Table 1, total of 16 strains of halotolerant microorganisms were isolated, 9 strains from the freshwater sample of Samrak Wetland Ecology Park, and 7 strains were isolated from Daejeo Ecological Park through this work. In addition, the marine agar medium, mainly composed of mineral salts, is a favorable medium for the cultivation of seawater-derived microorganisms. Confirmation of the potential industrial application of the strains was tested using complex media (nutrient agar, R2A agar, tryptic soy agar) that are being used for mass cultivation of the microorganisms. As a result, all 16 strains were able to grow (including weak growth) in at least one type of complex medium. Salt tolerance of the halotolerant strains was tested at 5% and 10% salt concentrations using marine agar medium (MA, 3% NaCl). As a result, it was confirmed that 8 strains (50%) were able to grow (including weak growth) under both 5% and 10% NaCl conditions, and 4 strains (25%) grew only under 3% NaCl conditions. Also, in order to confirm optimal pH conditions for growth, a marine agar medium adjusted at pH 5, 7, 9 was used. All isolated strains were able to grow at pH 7, and 10 strains (No. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) showed growth at pH 5 and pH 9 (Table 1).

Table 1. Isolation and identification of halophilic microorganisms isolated from the water of wetland in Korea

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The results of microbial identification using EzBioCloud's 16S-based ID analysis service on the basis of 16S rDNA nucleotide sequence of 16 aerobically isolated strains from domestic wetland freshwater samples were largely divided into 3 phyla, 3 classes, 4 orders, 5 families, 7 genera, and 13 species (Table 2), and the identification result of the isolated strains and their closest strains definition is shown in Table 3. In addition, a phylogenetic tree was constructed to confirm the relationship between the isolated and identified strains (Fig. 1). As shown in Table 2, Firmicutes (Bacilli) was dominant with 81.3%, Proteobacteria (Gammaproteobacteria) was next with 12.5%, and Actinobacteria (Actinomycetia) was the least with 6.2%. The 13 strains from phylum Firmicutes that dominated were distributed in two families Bacillaceae and Planoco- ccaceae, with 84.6% and 15.4%, respectively. From phylum Proteobacteria, only Gammaproteobacteria class species were isolated, and isolated strains belonged to the family Yersiniaceae and Aeromonaceae, each accounting 50%. Lastly, 1 strain belonged to the family Microbacteriaceae in the phylum Actinobacteria.

Table 2. Phylum analysis of halophilic microorganisms isolated from the water of wetland in Korea

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Table 3. Representative sequences of halophilic microorganisms isolated from the water of wetland in Korea

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Fig. 1. Evolutionary relationships of taxa by using the 16S rDNA sequences of bacteria from the wetland in Korea. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. This analysis involved 16 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1618 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

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Analysis of hydrolytic enzymes and auxin activity was performed for possible application of isolated halotolerant strains in the food and cosmeceutical industries and potential use as microbial fertilizer in the eco-friendly agricultural industry. As shown in Table 3, it was confirmed that all strains, except 3 strains, had at least one hydrolytic enzyme activity and 5 strains (Deposit number: NMC4-B510, NMC4-B512, NMC4-B513, NMC6-B436 & NMC4-B450) showed more than 2 enzyme activities. Weak auxin production was confirmed in 1 strain (Deposit number: NMC6-B436) isolated from the freshwater sample of Daejeo Ecological Park.

Duan and their colleagues conducted a characteristic study of β-amylases produced by the Bacillus aryabhattai GEL-09 strain and reported that they could be applied for starch hydrolysis (Duan et al., 2021). Therefore, it is expected to be applied to the food industry of Bacillus aryabhattai (Deposited number: NMC4-B512), which is one of the isolated strains with high amylase and protease activity in this study. Moreover, the antibacterial ability of Bacillus licheniformis SCK B11 against harmful bacteria and the characteristics of biogenic amine decomposition were reported (Kim et al., 2012), indicating that Bacillus licheniformis (Deposited number: NMC4-B513) also can be used as a candidate for manufacturing of traditionally fermented soybean products that are difficult to control pathogens and biogenic amine levels.

In addition, the halotolerant microorganisms isolated through this study will be of great significance in terms of securing the diversity of domestic microbial biological resources. The eight isolates from the wetland showed growth at 10% NaCl concentration and these isolates showed growth at pH 9 unlike other isolates from the same wetland sample. These 4 isolates (NMC4-B509, NMC4-B510, NMC4-B511 & NMC4-B454) showed highest protease activity corresponding to +++ (Table 1 & 3). Alkaline protease from these halotolerant strains that grow under high salt concentration conditions can be used not only for detergents but in food-grade products broadening its application range (Mayuri et al., 2019). Therefore, in this study, auxin production capacity analysis was carried out for use as a microbial fertilizer that replaces chemical fertilizers by promoting the growth of crops along with the activity of three enzymes (amylase, lipase, protease) applicable to starch processing, food industry, and industrial enzyme market for detergent using halophilic microorganisms isolated from domestic wetlands located close to the sea. It is expected that this will enhance the value of halophilic microbial resources derived from wetlands and be used as basic biological materials for industrial enzyme-related biotechnology and microbial fertilizer research (Jeong et al., 2021).

It is expected that it can be used as a basic biological material for enzyme-related biotechnology for application to the food and cosmetic industries as well as the possibility of discovering new strains. Also, all strains isolated through this study were deposited in the Microbial Value Enhancement Project, Korea Research Institute of Bioscience and Biotechnology.