Chiang Mai J. Sci. 2018; 45(4) 1595 Chiang Mai J. Sci. 2018; 45(4) : 1595-1609 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Isolation and Diversity of Actinomycetes from Sediments of Different Depths Between 34 m and 3,235 m in South China Sea Manita Kamjam [a], Qingyi Xie [b], Zixin Deng [a] and Kui Hong* [a] [a] Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, PR China. [b] Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, P. R. China. * Author for correspondence; e-mail: [email protected] Received: 8 September 2017 Accepted: 29 January 2018 ABSTRACT Diversity and isolation of actinomycetes from marine sediments collected from the South China Sea at depths ranging from 34 to 3,235 m were carried using dilution plate technique with heat and non-heat pretreatment on 9 isolation media, and plate stamping technique incubated on RH and M6 media at 28 °C and 10 °C. A total of 43 actinomycete strains were isolated from the different depth of marine sediments, among of which 32 were from the deep sea samples (1,645-3,235 m). Non-heat pretreatment was effective for the actinomycete isolation from deep sea sediment samples. RH and M6 were the more effective media regarding the number and diversity of isolates recovered. A higher percentage of actinomycete colonies (19.64%) were obtained by using plate stamping technique comparing to dilution plate method (5.29%). However, low-temperature incubation (10 °C) was also significantly effective on actinomycetes isolation from deep-sea samples with a higher percentage of the ratio actinomycetes to other total viable bacteria. Higher diversity of actinomycetes genera were found in deep-sea regions (depth 1,645 m-3,235 m) comparing to shallow regions marine sediments (depth <200 m). Partial 16S rRNA gene sequence data revealed that the isolates belong to the Class Actinobacteria, with genetic affiliations to five orders, six families, and six genera. Micromonospora (n= 23) and Streptomyces (n= 14) were the dominant genera, followed by Dietzia (n= 2), Tsukamurella (n=2), Blastococcus (n=1) and Microbacterium (n=1). Keywords: marine actinomycetes, isolation, diversity, deep sea 1. INTRODUCTION The vast oceans cover 70 % of the 2,000 and 6,000 m) and the hadal zone world’s surface, with 95% greater than (depths below 6,000 m) [1]. At about 200 m 1,000 m deep. Deep sea environments are depth, the deep sea is characterized by high divided into the bathyal zone (depths between pressure, low temperature, lack of light and 200 and 2,000 m), the abyssal (depths between variable salinity and oxygen concentration. 1596 Chiang Mai J. Sci. 2018; 45(4) Deep-sea organisms have developed unique [8, 9]. Some previous reports on the isolation biochemical metabolic and physiological and diversity of actinomycetes from the capabilities, which not only ensure their survival deep-sea environment showed that the in this habitat but also provide a potential for genus Micromonospora and Streptomyces were the production of novel metabolites absent dominant genera in the deep-sea. For example, in terrestrial microorganisms. The increasing Micromonospora was found to be dominant in numbers of literature on novel metabolites the Mediterranean (2,800 m and 4,400 m) and diversity of marine actinomycetes and Trondheim Fjord (Norway) [10, 11]. strongly supported the view that the marine In addition, Streptomycetes were reported environments including deep sea are to be dominant in deep-sea coral ecosystems significant source for search and discovery of Cantabrian Sea (1,500-4,700 m) [12], and of both diversity and novel secondary also presented in considerable number in metabolites [2, 3]. the Mariana Trench (10,898 m) [13]. Novel Several factors influence the actinomycete species isolated from deep sea actinomycetes isolation from marine environment between 2006 and 2016, have sediments, including heat pretreatment, yielded an impressive array of novel species composition of selective media and isolation including 21 species under 13 genera. The methods. Heat pretreatment procedures highest number of deep sea actinomycete have been effectively used for the selective was found at depths of the abyssal zone isolation of members of several actinomycete and the deeper regions, with the maximum taxa by decreasing the ratio of bacteria to number from Microbacterium, followed by actinomycetes on isolation plates, but these Dermacoccus, Streptomyces and Verrucosispora. procedures may also reduce the number Eight genera of actinomycetes were reported of actinomycetes [4]. Moreover, the isolation to produce secondary metabolites, among media were designed to favor the growth of which Streptomyces was the richest producer actinomycetes and reduce the development [14]. of unwanted microorganisms [5]. For The high number of novel actinomycete example, Vickers et al. [6] designed raffinose- species isolated from the deeper regions of histidine agar plates for the isolation of South China Sea showed a potentially rare streptomycetes by reducing the frequently abundant of novel genera/species. Their isolated species-group; plate stamping method secondary metabolites are remarkably equal was found to show good actinomycetes to that of Mariana Trench [2, 14]. However, recovery from marine sediments [7]. Thus, the few studies focused on the estimation of developments of efficient cultivation methods microbial diversity at different depths in the are important for better understanding of South China Sea. In this study, we report the actinomycetes diversity of deep-sea habitats diversity of culturable actinomycetes in the to obtain novel species or novel compounds. South China Sea marine sediment samples Microbial diversity in deep-sea regions from different depths (34 m - 3,235 m). could be remarkably high with species In addition, the dilution plate technique richness. The expanding of microbial with heat/non-heat pretreatment, selective biodiversity reach to the 5,000 m depth to media and plate stamping technique are the abyssal zone than the peak amount of compared for the selective isolation of species at the depths of 3,000 m and beyond actinomycetes. Chiang Mai J. Sci. 2018; 45(4) 1597 2. MATERIALS AND METHODS There are seven samples of “Non-deep sea” 2.1 Environmental Samples (depths ranging from 20 to 156 m), two Fourteen marine sediment samples were samples of “Bathyal” (depths ranging from collected from different locations in the 1,471 m and 1,645 m), and five samples South China Sea, at depths ranging from of ”Abyssal” (depths ranging from 2,017 34 m to 3,235 m (Table 1). Sediment sample to 3,235 m). All samples were preserved at depths are classified as “Non-deep sea”, 4 °C until they were processed for isolation “Bathyal (depths between 200-2,000 m)” and of actinomycetes. “Abyssal (depths between 2000-6000 m)”. Table 1. Source of sediment samples. Oceanic zone Depth pH Coordinates (m) longitude latitude Non-deep sea 34 7.83 114.38 E 22.10 N 35 8.04 114.74 E 22.23 N 62 7.87 114.78 E 21.91 N 85 7.85 114.91 E 21.60 N 96 7.27 110.00 E 18.00 N 140 6.69 118.35 E 22.27 N 156 6.23 110.50 E 18.00 N Bathyal 1,471 5.93 111.00 E 18.00 N 1,645 6.46 115.37 E 20.27 N Abyssal 2,017 5.96 111.50 E 18.00 N 2,093 5.70 113.00 E 18.00 N 2,441 6.69 112.00 E 18.00 N 3,004 6.46 118.00 E 20.00 N 3,235 5.75 114.00 E 18.00 N 2.2 Selective Isolation and Enumeration 0.1 ml suspensions from each dilution tube of Actinomycetes from Marine Sediments were spread on 9 selective isolation media, A total of fourteen sediment samples such as OA (oatmeal agar, ISP medium 3); were subjected to three different methods for oatmeal, 60 g; agar, 15 g; pH 7.2~7.4; isolation and enumeration of actinomycetes. GS1 (Gause modified medium 1); soluble (1) The dilution plate technique with heat/ starch, 20 g; K2HPO4, 0.5 g; KNO3, 1 g; ⋅ non heat pretreatment: 1 g of sediment MgSO4 7H2O, 0.5 g; agar, 20 g; pH7.4~7.6; was added to 9 ml of sterilized artificial sea SC (IM6); yeast extract, 4 g; malt extract, water solution (1%; Sigma, Germany), and 30 g; glucose, 4 g; agar, 18 g; pH 7.0~7.4; agitated on a shaker for 30 min at 150 rpm HL2 (IM8); glucose, 10 g; peptone, 5 g; to disperse bacterial propagule, followed by tryptone, 3 g; NaCl, 5 g; agar, 15 g; pH 7.0; heat-pretreatment at 50 °C in a water bath RH (Raffinose-histidine medium); raffinose, ⋅ for 10 minutes [15] or left untreated 10 g; L-histidine, 1g; MgSO4 7H2O, 0.5 g; ⋅ (non- heat pretreatment). Serial dilutions of FeSO4 7H2O, 0.01 g; K2HPO4, 1g; agar, soil suspensions up to 10-3 were made and 20.0 g; pH 7.0~7.4; HV (Humic acid -vitamin 1598 Chiang Mai J. Sci. 2018; 45(4) agar); humic acid, 1.0 g; CaCO3, 0.02 g; total genomic DNA samples from all the Na2HPO4, 0.5 g; KC1, 1.7 g; FeSO4.7H2O, actinomycetes isolates were extracted ⋅ ® 0.01 g; MgSO4 7H2O, 0.5 g; agar, 18 g; with Fast DNA SPIN Kit followed pH7.2; SIM (Starch casein agar); starch, 10 g; manufacturer’s protocol. The PCR reactions casein, 3 g; agar, 18 g; pH 7.0~7.4 [16], were performed in a final volume of 25 μl μ M6; glycerine, 6 ml; arginine, 1 g; K2HPO4, composed of DNA template (1 l upper ⋅ 1 g; MgSO4 7H2O, 0.5 g; pH 7.2~7.4 [17] aqueous layer), I-5TM 2X High-Fidelity and 1:100 Marine agar; yeast extract, 0.01 g; Master Mix (12.5 μl) and 20 μM of primer peptone, 0.05 g boric acid, 0.02 mg; NaCl, Eubac27F and primer Eubac1492R with 0.19 g; MgCl2, 0.09 g; Na2SO4, 0.03 g; CaCl2, the appropriate reaction buffer under 0.02 g; KCl, 5.50 mg; NaHCO3, 1.6 mg; the following conditions: initial denaturation ⋅ ° FeC6H5O7 3H2O, 1 g KBr, 0.80 mg; SrCl2, at 94 C for 5 min, followed by 32 cycles of ° ° 0.34 mg; Na2HPO4, 0.08 mg; Na2SiO3, 94 C for 30 s, annealing at 55 C for 30 s, ° 0.04 mg; NH4NO3, 0.016 mg; agar, 18 g; and 72 C for 90 s.