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Redalyc.Characterization of a Hyperthermophilic Sulphur-Oxidizing Electronic Journal of Biotechnology E-ISSN: 0717-3458 [email protected] Pontificia Universidad Católica de Valparaíso Chile Valdebenito-Rolack, Emky; Ruiz-Tagle, Nathaly; Abarzúa, Leslie; Aroca, Germán; Urrutia, Homero Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring Electronic Journal of Biotechnology, vol. 25, 2017, pp. 58-63 Pontificia Universidad Católica de Valparaíso Valparaíso, Chile Available in: http://www.redalyc.org/articulo.oa?id=173349762012 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Electronic Journal of Biotechnology 25 (2017) 58–63 Contents lists available at ScienceDirect Electronic Journal of Biotechnology Research article Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring Emky Valdebenito-Rolack a,⁎, Nathaly Ruiz-Tagle a, Leslie Abarzúa a,GermánArocab,HomeroUrrutiaa a Laboratorio de Biopelículas y Microbiología Ambiental, Centro de Biotecnología, Universidad de Concepción, Concepción, Chile b Escuela de Ingeniería Bioquímica, Facultad de Ingeniería, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile article info abstract Article history: Background: Sulphur-oxidizing microorganisms are widely used in the biofiltration of total reduced sulphur Received 18 August 2016 compounds (odorous and neurotoxic) produced by industries such as the cellulose and petrochemical Accepted 10 November 2016 industries, which include high-temperature process steps. Some hyperthermophilic microorganisms have the Available online 24 November 2016 capability to oxidize these compounds at high temperatures (N60°C), and archaea of this group, for example, Sulfolobus metallicus, are commonly used in biofiltration technology. Keywords: Results: In this study, a hyperthermophilic sulphur-oxidizing strain of archaea was isolated from a hot spring Biofilms on polyethylene (Chillán, Chile) and designated as M1. It was identified as archaea of the genus Sulfolobus (99% homology Biofiltration fi Cellulose industries with S. solfataricus 16S rDNA). Bio lms of this culture grown on polyethylene rings showed an elemental -1 -1 Denaturing gradient gel electrophoresis sulphur oxidation rate of 95.15 ± 15.39 mg S l d , higher than the rate exhibited by the biofilm of the extremophile sulphur-oxidizing archaea S. metallicus (56.8 ± 10.91 mg l-1 d-1). Hyperthermophile Conclusions: The results suggest that the culture M1 is useful for the biofiltration of total reduced sulphur gases at Industrial gas emissions high temperatures and for other biotechnological applications. Petroleum refinery Sulfolobus © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. Sulphide This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Sulphur-oxidizing archaea Sulphur-oxidizing microorganisms 1. Introduction a mesophilic reactor because no additional cooling equipment is necessary [8]. Few reports have been published about biofiltration The use of sulphur-oxidizing bacteria (SOB) has been proposed to under thermophilic conditions [14], and only four of them describe oxidize total reduced sulphur compounds (TRS) present in industrial TRS biodegradation [7,12,15,16]. However, there are several reports gas emissions [1,2,3]. These gaseous compounds, emitted by several that describe microbial communities and enriched consortia from hot industrial processes, are toxic [4] and odorous [5].Theuseofbiofilters springs composed mainly of chemolithotrophic archaea from the inoculated with SOB has been shown to be a good solution for treating genera Sulfolobus, Acidianus,andMetallosphaera [17,18]. In fact, all theseemissionsatmoderatetemperatures, but there are many gaseous known sulphur-oxidizing extreme hyperthermophiles (optimum emissions from industrial processes containing these compounds at temperature N 60°C) are crenarchaeotes [19]. Some hyperthermophilic high temperatures (N50°C), especially in boiler combustion, petroleum sulphur-oxidizing prokaryote cultures have been isolated, predominated refinery, smelting and composting facilities [6,7] because most by archaea, for example, the VS2 culture found in hot spring sediments biochemical transformations occur more rapidly at high temperature [8]. from underground mines at Hokkaido, Japan [18], which can be used To date, most of the studies reporting TRS oxidation using SOB in bioleaching. Another example is found in the hyperthermophilic biofilms involve mesophilic or moderate thermophilic conditions microorganisms characterized in geysers in the Yellowstone National [9,10,11,12]. The immobilization of thermophilic desulphurisation Park, USA [17]. In these reports, the main archaea genera were prokaryotes on a packing support material in a bioreactor operating Sulfolobus, Metalosphaera,andThermoplasma; however, the latter is under thermophilic conditions produces more rapid and economical mainly heterotrophic. In the first study mentioned above, the optimum treatment processes [13]. The advantages of using a thermophilic temperature for S0 (elemental sulphur) oxidation was 70°C in a culture bioreactor are high degradation kinetics and lower cost than dominated by Sulfolobus metallicus and Thermoplasma acidophilum,with S0 oxidation rate of 99 mg S0 L-1 d-1. One of the most frequently studied genera of hyperthermophilic sulphur-oxidizing archaea is Sulfolobus, ⁎ Corresponding author. E-mail address: [email protected] (E. Valdebenito-Rolack). which contains widely described species like S. metallicus and Sulfolobus Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. solfataricus. http://dx.doi.org/10.1016/j.ejbt.2016.11.005 0717-3458/© 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). E. Valdebenito-Rolack et al. / Electronic Journal of Biotechnology 25 (2017) 58–63 59 To date, no S0-oxidizing microorganisms from volcanic thermal epifluorescence microscopy with DAPI (4′,6-diamidino-2-phenylindole) environments have been described; therefore, the study of the S0 stain. oxidizing efficiency of a biofilm of hyperthermophilic prokaryotes from such environment is of great interest for the application in TRS 2.5. Sulphur oxidation rate biofiltration under thermophilic conditions. The present study aimed to characterize a microbial community At the end of the 30 d incubation period, the culture medium obtained from a hot spring located in the Andes Mountains near was discarded and made up with fresh 9 K liquid culture medium, Chillán, Chile, by determining the oxidation rate of S0 in comparison with the addition of 10 g L-1 S0 as energy source. The S0 oxidation rate with that achieved by a biofilm of S. metallicus.Themicrobial was monitored for 30 additional days to measure the sulphate cultures were characterized using 16S rDNA profile, sequencing and concentration as the final product of the S0 bio-oxidation process, phylogenetic analysis. assuming that 71 mg S0 L-1 d-1 is equivalent to 214 mg L-1 d-1 -2 -2 de SO4 [18].TheSO4 concentration was measured by BaSO4 2. Materials and methods spectrophotometry at 420 nm using a SulfaVer 4 kit and following the manufacturer's instructions (HACH LANGE, Dusseldorf, Germany). The -1 2.1. Samples measurements were performed every 7 d, and a sulphate (mg L ) vs, time (d) plot was made. The sulphate oxidation rate was calculated by Three samples of sediment (5-g wet weight, taken using a sterile linear regression using the software Prim 6.0 (GraphPad Software steel spoon), and water (40 mL, taken using a sterile plastic 50-mL Inc.). In this phase, growth was controlled as described in Section 2.5. tube) were taken from a hot spring located in the Andes Mountains All the experiments were conducted with the M1 strain and the near Chillán (36° 54′ 35″S/71° 25′ 4″W, 80°C and pH = 3), as standard strain, including a negative control, in triplicate. described by Coram-Uliana et al. [20], and transported to the laboratory where they were stored at 4°C in darkness. 2.6. Analysis of results For biofilm development and sulphur oxidation rates, the values are 2.2. Standard bacterial strains expressed as the mean of three replicates. Growth differences and oxidation rate comparisons were made by measuring the significance S. metallicus DSMZ 6482 was used as standard positive control in S0 of the difference between the averages of the parameters mentioned, degradation and for molecular characterization experiments. using one-way ANOVA (α ≤ 0.05). 2.3. Microbial enrichment 2.7. DNA extraction fl Cultures were performed in 500-mL Erlenmeyer asks with Pellets were collected from 1.6 mL of an active culture or an original fi hermetic stopper, using a 9 K modi ed culture medium (Table 1), sample diluted by centrifugation in Eppendorf tubes at 8000 rpm for -1 0 with 10 g L of S as energy source, and incubated on a shaker 3 min. The supernatant was eliminated, and each pellet was – (170 rpm) at 60 80°C [7,18,21]. The cultures were transferred to new re-suspended in 310 μL of HTE buffer (HTE: 50 mM Tris–HCl, 20 mM fl asks with fresh medium
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