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Bacterial Composition and Diversity in Deep-Sea Sediments from the Southern Colombian Caribbean Sea

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Bacterial Composition and Diversity in Deep-Sea Sediments from the Southern Colombian Caribbean Sea diversity Article Bacterial Composition and Diversity in Deep-Sea Sediments from the Southern Colombian Caribbean Sea Nelson Rivera Franco 1 , Miguel Ángel Giraldo 1,2, Diana López-Alvarez 1,* , Jenny Johana Gallo-Franco 3, Luisa F. Dueñas 4,5 , Vladimir Puentes 4 and Andrés Castillo 1,2,* 1 TAO-Lab, Centre for Bioinformatics and Photonics-CIBioFi, Universidad del Valle, Calle 13 # 100-00, Edif. E20, No. 1069, Cali 760032, Colombia; [email protected] (N.R.F.); [email protected] (M.Á.G.) 2 Department of Biology, Universidad del Valle, Calle 13 No 100-00, Edif. E20, Cali 760032, Colombia 3 Natural Sciences and Mathematics Department, Pontificia Universidad Javeriana-Cali, Cali 760032, Colombia; [email protected] 4 Anadarko Colombia Company-HSE, Calle 113 No. 7-80 Piso 11, Bogotá D.C. 110111, Colombia; [email protected] (L.F.D.); [email protected] (V.P.) 5 Department of Biology, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 No. 26-85, Bogotá D.C. 111321, Colombia * Correspondence: [email protected] (D.L.-A.); [email protected] (A.C.) Abstract: Deep-sea sediments are considered an extreme environment due to high atmospheric pressure and low temperatures, harboring novel microorganisms. To explore marine bacterial diversity in the southern Colombian Caribbean Sea, this study used 16S ribosomal RNA (rRNA) gene sequencing to estimate bacterial composition and diversity of six samples collected at different depths (1681 to 2409 m) in two localities (CCS_A and CCS_B). We found 1842 operational taxonomic units (OTUs) assigned to bacteria. The most abundant phylum was Proteobacteria (54.74%), followed by Bacteroidetes (24.36%) and Firmicutes (9.48%). Actinobacteria and Chloroflexi were also identified, but their dominance varied between samples. At the class-level, Alphaproteobacteria was most Citation: Franco, N.R.; Giraldo, M.Á.; abundant (28.4%), followed by Gammaproteobacteria (24.44%) and Flavobacteria (16.97%). The López-Alvarez, D.; Gallo-Franco, J.J.; results demonstrated that some bacteria were common to all sample sites, whereas other bacteria were Dueñas, L.F.; Puentes, V.; Castillo, A. unique to specific samples. The dominant species was Erythrobacter citreus, followed by Gramella sp. Bacterial Composition and Diversity Overall, we found that, in deeper marine sediments (e.g., locality CCS_B), the bacterial alpha diversity in Deep-Sea Sediments from the decreased while the dominance of several genera increased; moreover, for locality CCS_A, our results Southern Colombian Caribbean Sea. suggest that the bacterial diversity could be associated with total organic carbon content. We conclude Diversity 2021, 13, 10. https://doi. that physicochemical properties (e.g., organic matter content) create a unique environment and play org/10.3390/d13010010 an important role in shaping bacterial communities and their diversity. Received: 16 October 2020 Accepted: 18 December 2020 Keywords: bacteria; 16S rRNA; metabarcoding; marine sediments; depth Published: 31 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims 1. Introduction in published maps and institutional Most microorganisms have specific habitat requirements, and many might be found affiliations. only in some regions of the planet, particularly areas which have not been widely explored by humans due to limited accessibility. Perhaps one of the most challenging environments to access is the deep sea, which is among the least studied natural habitats and the most Copyright: © 2020 by the authors. Li- extended in the planet. The deep sea combines unique conditions, such as temperatures ◦ censee MDPI, Basel, Switzerland. This below 4 C, lack of light, and high hydrostatic pressure [1]. It has been estimated that article is an open access article distributed marine subsurface habitats harbor majority of the global prokaryotic biomass [2]. These under the terms and conditions of the conditions can promote the presence of microbial communities with specific features and Creative Commons Attribution (CC BY) requirements, which make them unculturable and difficult to study. It has even been license (https://creativecommons.org/ reported that environments with different properties, separated by a few kilometers, can licenses/by/4.0/). exhibit different microbial communities [3,4]. Currently, metabarcoding techniques allow Diversity 2021, 13, 10. https://doi.org/10.3390/d13010010 https://www.mdpi.com/journal/diversity Diversity 2021, 13, 10 2 of 13 culturable and nonculturable microorganisms to be identified through 16S ribosomal RNA (rRNA) gene sequencing using high-throughput sequencing (HTS) technology. This includes identifying the taxonomic diversity and community composition of bacteria and archaea domains. The 16S rRNA gene has ubiquitous distribution across all archaeal and bacterial lineages, is evolutionarily conserved, and contains many variable regions that facilitate its use [5]. Besides, it is widely available in public databases (e.g., RDP (http://rdp.cme.msu.edu/), SILVA [6], and Greengenes [7,8]) as a result of the deployment of HTS technology. Molecular tools have been widely used to conduct crucial global research on metage- nomics in marine environments. Large-scale expeditions have been carried out for ocean sampling worldwide, such as the Global Ocean Sampling expedition, which collected ocean water samples from 23 countries and four continents, and the Tara Ocean Foundation expe- dition in 2003, which obtained nearly 35,000 shallow ocean samples [9,10].There have been several studies evaluating the diversity and distribution of bacteria in deep-sea surface sediments. For example, in the South Atlantic Ocean, 17 samples were analyzed across depths ranging from 5 to 2700 m [11]; in the Pacific Ocean, four samples were studied from Arctic sediments [12]; in the southeastern Atlantic Ocean, five samples were collected from the Cape, Angola, and Guinea Basins at depths ranging from 5032 to 5649 m [3]; in the South Atlantic Polar Front, 15 samples were analyzed across a depth range of 3655 to 4869 m [13]; and in North Sao Paulo Plateau, five samples were collected between 2456 and 2728 m of depth [14]. In Colombia, there have been no studies yet on bacterial communities in deep-sea sediments of the Caribbean Sea. However, some aquatic metage- nomic studies have been conducted, for example, research on acidic hot springs in the Colombian Andes [15] and studies analyzing microbiomes associated with corals, algae, sponges, and shallow-water sediment [16–19]. The southern Colombian Caribbean is a tectonically active, convergent margin with high sediment supply [20] and is probably the main source of nutrients supporting biological productivity in the oligotrophic Caribbean Sea [21]. Therefore, this region could harbor a diversity of microorganisms that have not been studied until now. In this study, we investigated the bacterial composition and diversity in deep-sea sediments at two sites of the southern Colombian Caribbean Sea as well as the relationship between bacterial communities in marine sediments and the effect of six different depths and physicochemical properties. 2. Materials and Methods 2.1. Sampling Between October and November of 2018, we collected six deep-sea sediment sam- ples at depths ranging from 1681 to 2409 m in two localities in the southern Colombian Caribbean Sea (Figure1, Table1, and Table S1). We obtained three 0.25 m 2 samples per locality using a box corer. The sediment samples were stored at −80 ◦C until they were processed for DNA isolation. Abiotic analyses were performed to measure physicochemi- cal properties, such as granulometry, total organic carbon (TOC), and iron content. The properties at the two sampling sites differed with regard to their bulk parameters. All the samples were dominated by silt and clay (Table1), except for sample CCS_B1, which was dominated by gravel and sand. The iron concentration increased with depth, while %TOC decreased, except for CCS_B. According to the literature, the salt concentration in the South Caribbean coast at a depth of around of 1000 m is approximately 35 psu, while the temperature is 0 to 5 ◦C[22,23]. Diversity 2021, 13, 10 3 of 13 Figure 1. Geographic location of the two deep-sea sampling localities in the southern Colombian Caribbean Sea: three samples from locality CCS_A (red dots) and three samples from locality CCS_B (blue dots); Table S1. Table 1. Metadata of deep-sea sediment samples collected at the southern Colombian Caribbean Sea. Sample- Depth %TOC Iron ID Locality (m) %Gravel %Sand %Silt %Clay (mg/Kg) CCS_A1 CCS_A 1681 0.39 4.80 42.03 52.78 7.04 29,900 CCS_A2 CCS_A 1814 0.17 3.55 40.72 55.56 7.06 30,600 CCS_A3 CCS_A 1875 0.19 3.55 42.21 54.05 6.15 31,300 CCS_B1 CCS_B 2221 1.50 7.41 37.85 53.24 6.35 26,600 CCS_B2 CCS_B 2397 0.04 2.66 39.41 57.89 5.85 38,100 CCS_B3 CCS_B 2409 0.20 2.40 48.23 49.17 5.50 38,600 2.2. DNA Isolation and 16S rRNA Gene Sequencing Total DNA was isolated from 1 g of sediment (homogenized subsample of the top 5 cm collected) for each sample using the DNeasy Powersoil Kit (Qiagen, Hilden, Alemania), according to the manufacturer’s instructions. DNA quality was evaluated by agarose gel electrophoresis and PicoGreen method using a Victor 3 fluorometer. Six barcoding libraries were constructed for the variable V3–V4 region of the 16S rRNA gene using the Nextera XT Index Kit V2 with primers Bakt_341F: CCT ACG GGN GGC WGC AG and Bakt_805R: GAC TAC HVG GGT ATC TAA TCC [24]. All PCR reactions were performed in total reaction volumes of 25 µL containing KAPAHiFi HotStart Ready Mix (2X) (Sigma-Aldrich, St. Louis, MO, USA), 1 µM of forward and reverse primers, and 5 ng/µL template DNA suspended in 10 mM Tris pH 8.5. The thermal cycling profile consisted of initial denaturation at 95 ◦C for 3 min, followed by 25 cycles of denaturation at 95 ◦C for 30 s, annealing at 55 ◦C for 30 s, elongation at 72 ◦C for 30 s, and a final cycle at 72 ◦C for 5 min.
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