Bozo-Hurtado et al. Aquatic Biosystems 2013, 9:17 http://www.aquaticbiosystems.org/content/9/1/17 AQUATIC BIOSYSTEMS RESEARCH Open Access Identification of bacteria in enrichment cultures of sulfate reducers in the Cariaco Basin water column employing Denaturing Gradient Gel Electrophoresis of 16S ribosomal RNA gene fragments Lorelei Bozo-Hurtado1, M Alexandra García-Amado2, Andrei Chistoserdov3, Ramon Varela4, J Jesus Narvaez4, Rita Colwell5 and Paula Suárez1* Abstract Background: The Cariaco Basin is characterized by pronounced and predictable vertical layering of microbial communities dominated by reduced sulfur species at and below the redox transition zone. Marine water samples were collected in May, 2005 and 2006, at the sampling stations A (10°30′ N, 64°40′ W), B (10°40′ N, 64°45′ W) and D (10°43’N, 64°32’W) from different depths, including surface, redox interface, and anoxic zones. In order to enrich for sulfate reducing bacteria (SRB), water samples were inoculated into anaerobic media amended with lactate or acetate as carbon source. To analyze the composition of enrichment cultures, we performed DNA extraction, PCR-DGGE, and sequencing of selected bands. Results: DGGE results indicate that many bacterial genera were present that are associated with the sulfur cycle, including Desulfovibrio spp., as well as heterotrophs belonging to Vibrio, Enterobacter, Shewanella, Fusobacterium, Marinifilum, Mariniliabilia,andSpirochaeta. These bacterial populations are related to sulfur coupling and carbon cycles in an environment of variable redox conditions and oxygen availability. Conclusions: In our studies, we found an association of SRB-like Desulfovibrio with Vibrio species and other genera that have a previously defined relevant role in sulfur transformation and coupling of carbon and sulfur cycles in an environment where there are variable redox conditions and oxygen availability. This study provides new information about microbial species that were culturable on media for SRB at anaerobic conditions at several locations and water depths in the Cariaco Basin. Keywords: SRB, Cariaco Basin, Desulfovibrio, Vibrio, DGGE, Culture Background Based on redox conditions and oxygen content, the basin The Cariaco system is a depression located on the northern is divided into three layers: oxic (~ 0-250 m); redox transi- continental shelf of Venezuela in the Caribbean Sea and is tion (~ 250–450 m); and anoxic (~ 450 to 1400 m) [1-4]. largest true marine permanently anoxic marine water body The basin water column is characterized by a pronounced in the world. The Basin, 160 km long and 50 km wide, is vertical layering of microbial communities. The oxic divided into two sub-basins, each with a maximum depth layer possesses the most complex trophic structure, of 1400 m and separated by a saddle at 900 m water depth. dominated by processes such as photosynthesis, aer- obic heterotrophy and nitrification. The redox transi- * Correspondence: [email protected] tion zone is biogeochemically stratified, appears less 1Departamento de Biología de Organismos, Universidad Simón Bolívar, complex and predominant processes are chemoautotrophy, Caracas, Venezuela fermentation, denitrification, and anaerobic respiration. Full list of author information is available at the end of the article © 2013 Bozo-Hurtado et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bozo-Hurtado et al. Aquatic Biosystems 2013, 9:17 Page 2 of 11 http://www.aquaticbiosystems.org/content/9/1/17 Theanoxiczonepresumablyhasthesimplesttrophic explore the diversity of bacteria in the Cariaco Basin in- structure that appears to be supported by fermentation, volved in sulfate reduction, we used SRB enrichment cul- sulfate reduction, methanogenesis and anaerobic methane ture complemented with identification of the enriched oxidation [3]. Many studies have been conducted in the bacteria by gradient gel electrophoresis (DGGE). Cariaco Basin to understand how microorganisms are dis- tributed in the stratified water column environment and Results how they influence geochemical processes [1,3,5]. Interest- Depth profiles of temperature, salinity, and dissolved ingly, high levels of sulfide present in the Cariaco Basin oxygen in the water column at all stations for two years have been attributed to biological sulfate reduction [6]. are shown in Table 1. Temperatures varied from 17.10 This is the first attempt to identify bacteria related with to 23.85°C, with the greater variation between the sur- sulfate reduction using enrichments cultures for the face and interface zone. Salinity was stable throughout Cariaco Basin. the water column at all depths and stations sampled, Several studies have been published describing the mi- ranging between 36.26 to 36.88 PSU. Dissolved O2 con- crobial community associated with the Cariaco Basin sulfur centration peaked at 40 m (4.009 mL/L) and declined cycle. Tuttle and Jannasch (1973) isolated several sulfide dramatically at depths below 200 m. The maximum mea- and thiosulfate-oxidizing bacteria and Morris et al. (1985) sured sulfide concentration was obtained at the greatest isolated Alteromonas sp. from thiosulfate-containing en- depth for all stations during both years of sampling. These richment cultures [7,8]. With the development of molecu- physicochemical patterns are typical of the Cariaco Basin, lar biology, culture-independent methods have been used showing redox zonation and similar to those reported by to detect SRB populations in the Cariaco Basin [1,5,9]. To other authors [2,4,6-12]. Table 1 Physico-chemical parameters measured in the Cariaco Basin during the study Year 2005 Station Zone Depth (m) Temperature (°C) Salinity (PSU) Dissolved O2 (mL/L) *H2S(μM) A Oxic 230 17.90 36.43 0.0738† ND Interface 270 17.69 36.39 0.0688 1.22 Anoxic 900 17.03 36.26 0.0653 51.47 B Oxic 235 17.83 36.42 0.0711† ND Interface 275 17.63 36.38 0.0674 0.69 Anoxic 670 17.06 36.27 0.0646 ND Year 2006 Station Zone Depth (m) Temperature (°C) Salinity (PSU) Dissolved O2 (mL/L) *H2S(μM) A Oxic 100 21.08 36.79 3.2116 ND Interface 300 17.64 36.36 0.0157 5.39 Anoxic 400 17.46 36.33 0.0113 14.3 500 17.23 36.29 0.0113 25.55 B Oxic 215 17.97 36.41 0.0245† ND Interface 260 17.75 36.38 0.0122 6.62 290 17.70 36.37 0.0129 7.07 Anoxic 325 17.57 36.35 0.0131 11.09 640 17.10 36.26 0.0186 38.46 D Oxic 40 23.85 36.88 4.0087 ND 180 18.18 36.45 0.5738 ND Interface 270 17.80 36.39 0.0135 0.21 Anoxic 365 17.52 36.34 0.0197 13.31 500 17.40 36.33 0.0196 18.86 *Data from http://www.imars.usf.edu/CAR/. † Low DO values in oxic zones over the limit of interface layer where DO decays and H2S increases. PSU: Practical Salinity Units. ND: Not determined. Bozo-Hurtado et al. Aquatic Biosystems 2013, 9:17 Page 3 of 11 http://www.aquaticbiosystems.org/content/9/1/17 Bacteria enrichments in SRB media two from station B and four from station D (Figure 1). All Twenty-four cultures, in which black coloration with fer- of the cultures had variable cell morphology, with curved rous sulfide precipitation was observed, were selected for rod-shaped bacteria with polar spores predominant. further investigation. Using TWIN pack medium, two cul- tures were obtained during 2005: one from station A and Molecular identification another from station B. Using TP medium, we obtained All 16S RNA gene amplicons from the 24 cultures were thirteen cultures during 2006: one from station A, six from separated in the DGGE gels (Figure 1). The patterns com- station B, and six from station D. Using API medium, nine prised several bands, suggesting that different bacterial cultures were obtained during 2006: three from station A, types were present. Sixty-seven DGGE bands were excised A B2 API (290 m) API B2 B4 TP (640 m) B4 D7 API (365 m) D7 API B3.2 TwinB3.2 (235 m) (270 m) D2 API M D3 TP (305 m) A10 API (900 m) A10 API A9 API (500 m) A9 API D5 TP (40 m) B8 TP (245 m) B8 (500 m) D4 API M A3 API (300 m) A3 API 1 M1 11 M2 8 9 6 17 M3 19 13 M4 15 M5 B D8 TP (400 m) B3 TP (325 m) B3 A3.1 Twin (230 m) B1 API (260 m) API B1 B7 TP (215 m) B7 B1 TP (260 m) B1 A8 TP (400 m) B2 TP (290 m) B2 (40 m) D5 API D2 TP (270 m) D7 TP (365 m) D4 TP (500 m) M M 59 39 49 51 M2 57 41 46 63 67 40 53 55 58 M3 M4 52 M5 Figure 1 16S RNA gene amplified from DNA isolated from cultures. Separation of PCR products amplified with bacteria specific 16S rRNA primers in 6% polyacrylamide gels with 0–100% denaturing gradient. Panels A and B. 16S RNA gene amplified from DNA isolated from 24 cultures. The number above each lane indicates the culture name and the number in parenthesis represents depth of isolation in meters. Lanes M are the ladders: M1= Aquimarina muelleri, M2= Escherichia coli, M3= Sulfurimonas denitrificans, M4= Vibrio alginolyticus and M5= Mycobacterium smegmatis. The sequenced bands are identified by number. Bozo-Hurtado et al. Aquatic Biosystems 2013, 9:17 Page 4 of 11 http://www.aquaticbiosystems.org/content/9/1/17 and sequenced, but only 23 quality sequences were acladewithV.