Description of Sampling Site: Salar de Punta Negra is a 250 Km2 athalassohaline wetland located in the Atacama Desert, northern Chile (24o 28' S, 69o 53' W, at 2,976 m.a.s.l.). It consists of several temporary saline lagoons covering about 3 Km2. Mining activities occurring around this place obtain most of the water for their processes from the groundwater below the salt flat through a network of extraction wells. The geographic and climatic characteristics of the Salar together with their chemical composition provide ideal conditions for the growth of extreme microorganisms, such as bacteria, archaea, and microalgae [35], which are the trophic bases of a unique aquatic flora and fauna (microalgae, microcrustaceae, rotifers, and flamingos). Water and sediment samples from 2 lagoons located in the eastern border of the Salar were collected using sterile spatulas and sterile plastic receptacles. Samples were transported in cooler boxers and processed immediately at the Biochemistry Laboratory of Universidad Católica del Norte.
Growth Media and Bacterial Isolation: Samples from the Salar were incubated in different media with and without sodium meta-arsenite (NaAsO2) in order to isolate and grow microorganisms. These media and their composition were named as Isolation Media (IM), Control Isolation Media (CIM), Growth Media Supplemented with As III (GM/S), and Growth Media Non Supplemented with As III (GM/NS), respectively, as shown in Table 2. A strain of Agrobacterium tumefaciens C58/ATCC 33970 was cultured in Luria broth and employed as positive control for arsenite oxidase detection. It was named Luria Media (LM). In order to promote the adaptation of isolated microorganisms and obtain a representative fraction of the microbial biodiversity in each sample, the isolation medium was prepared with sterile-filtered water from Salar de Punta Negra (SWS). This was done so as to make bacterial growth possible and maintain the chemical equilibrium present in the water from Salar de Punta Negra in these media. Thus, the nutritive agar (DIFCO) was dissolved in a minimal milli-Q water volume sterilized at 121°C/1 atm. Then, the volume was completed with SWS at 60°C. Table 2 shows the details of media composition. To obtain GM/S and GM/NS, growth media were prepared with a basis of Peptone Broth (DIFCO) supplemented and non-supplemented with NaAsO2.H2O (Merk), respectively. For GM/S preparation a sterile-filtered NaAsO2.H2O solution was added at 60°C, after autoclaving the media. The resulting concentration in the GM/S was 100 ppm in As III, prepared from a stock solution of NaAsO2.H2O 200 ppm.
Given the pKa of As(OH)3 (9.3) and the neutral pH of culture media, this compound dissociates according to equation 1, with an oxidation state 3+:
(1)
A mixture of surface sediments and water from Salar de Punta Negra was diluted in SWS in a 1:1000 ratio. This diluted mixture was named as dilute sample. Ten plates containing IM were inoculated with 1 mL of the previous dilute sample and incubated at 30°C until the presence of colonies, named here as “generation zero” (G0), was verified. Then, colonies from this generation, with different morphology (color, edge, and height), were transferred to plates with GM/S and incubated at 30°C for 18 to 24 h. The colonies obtained in this step were named as G1 and compared with the colonies of G0, through a Gram test. To increase the biomass required for the next experiments, colonies from G1 were transferred to flasks with 200 mL of GM/S until obtaining a new bacteria generation(G2).
Molecular Identification of Isolates: In order to determine the genomic variability between bacterial colonies, a restriction fragment length polymorphism analysis (RFLP) was conducted. Genomic DNA from isolates were obtained by Power Soil DNA isolation Kit (MoBio Laboratories Inc., Solana Beach, CA, US) and amplified by PCR. 16S rRNA genes were amplified using the primer pairs specific for Bacteria 8F (5′ AGAGTTTGATCCTGGCTCAG 3′) and 1392R (5′ ACGGGCGGTGTGTAC 3′), using 10 ng of DNA as a template in each amplification reaction. PCR conditions were selected as previously described by Norton et al. (2008) [35]. About ~1,500 bp amplicons were digested with HhaI and MspI restriction enzymes in separated reactions for 3 h at 37°C. Restriction patterns were compared by electrophoresis in 2% agarose gel and stained with etidium bromide.
Generational Cultures: To determine both, the presence of the arsenite-oxidase enzyme in bacterial lysates and if it was inducible in the presence of As (III), clones selected by RFLP analysis were cultured for several generations in media with and without NaAsO2. Thus, bacterial pellets of each of the seven isolates selected from G2 (10 μL) were inoculated in 200 mL of GM/S and GM/NS media (Table 2) and incubated for 18 h at 30°C to increase the biomass (G3). This procedure was repeated 10 times for each isolate to reach generation number 13th or G13. At the same time, the reference strain of Agrobacterium tumefaciens C58/ATCC 33970 was grown in LM, according to the protocol described above (Table 2). This bacterium was used for the Dot Blot experiments as a positive control for the expression of arsenite oxidase [36]. Finally, Bradford Analysis [37] was performed for total protein quantification from each isolate of G13.
Arsenic Kinetic Experiments: In order to determine the correlation between bacterial growth and As (III) removal from the culture media, the kinetics of 7 isolates from G13 was performed in separate experiments. Thus, 10 μL of pellets from each bacterial isolate were inoculated in screw cap tubes with 15 mL of GM/S and incubated at 30°C for 18 h. Once bacterial growth was obtained, 100 μL of bacterial inoculation were transferred to Erlenmeyer flasks containing 200 mL of GM/S and incubated at 30°C, while stirring at 300 rpm for 36 h. Medium controls consisting of 200 mL non-inoculated GM/S were incubated together with the previous experimental treatment flasks under the experimental conditions above to monitor the concentration of As (III) in the supernatants of the treatment flasks, relative to the total As. The latter is represented by arsenite concentration in the control medium since it does not suffer any significant change during the experiments.
As (III) in the culture medium of each treatment flask and the medium control were measured after bacteria removal, at specific incubation times: t0, t2, t4, t6, t8, t18, t22, t27, t30, and t36. To do this, 10 mL from each culture flask were filtered through sterile disposable filters of 0.22 μm pore. The filtered-recovered medium was used for arsenic measurements. The same procedure was used for the control culture flasks. Both, treatment and control flasks, were analyzed in triplicate. Simultaneously with arsenic measurements, non-filtered samples from the treatment flasks were used to estimate bacterial density. For this purpose, the samples were fixed in buffered glutaraldehyde (2% glutaraldehyde solution in 0.05 M phosphate buffer at pH 7.2-7.4) and cell counts were determined by the Neubauer chambers technique.
The arsenite removal capabilities of bacteria were expressed as Percentage of Arsenite Removal (PAR) and calculated from the initial values of As (III) present in the culture media (t0) and their corresponding values at each incubation time.
Arsenite/Total-Arsenic Quantification: Total-As and As (III) measurements were carried out by hydride-generation flow-injection atomic absorption spectrophotometry (FIAS-HG-AAS) with a detection limit of 0.001 ppm. This is a suitable method for the analysis of arsenic in waters, and it is used in most laboratories because of its high sensitivity, speed of analysis, and comparatively low cost [38, 39]. A basic validation analysis was applied to the data by calculating Reproducibility and Repeatability limits [40].
Arsenite Oxidase Dot Blot Analysis: A dot blot analysis was conducted for arsenite oxidase detection. Bacterial pellets of G13 at exponential growth phase were re-suspended in 4 mL of sterilized Milli-Q Water and lysed by sonication (140 Hz) with 3 30-s cycles on ice. Once the bacterial pellets were lysated, suspensions were transferred to 10 mL sterilized tubes. Then, 20 μL of 3 different protease inhibitors were added: aprotin (1 mg/mL); leupeptine (1 mg/mL); and PMSF/DMSO (100 mM). Five μL of each lysated were put on a nitrate-cellulose membrane (pore size of 0.47 mm Millipore). To check the specificity of antibodies and prevent cross reactivity, another 5 μL of bovine xantine oxidase (another molibdopterin protein) from bovine milk, Sigma – Aldrich diluted in 1:50, were dotted on the same membrane. This enzyme was chosen because it exhibits a high structural similarity with arsenite oxydase [14]. Arsenite oxydase purified from A. faecalis NCBI 8687 (792 Ao, 55 μM) [6], provided by Dr. Gretchen Anderson (Department of Chemistry, Indiana University, South Bend in USA), and lysates of A. tumefaciens C58/ATCC 33970 were used as positive controls of AOX expression. Sterilized GM/S was used as negative control for the same purpose. The membrane was blocked with 6% non-fat milk in TBS (0.1% Tween 20) (TBS-T) for 2 h at room temperature. To detect the arsenite oxidase, the membrane was incubated overnight at 4°C with diluted 1:25 in PBS-T polyclonal rabbit anti A. faecalis AOX primary antibody (793 ARs, 100 μg/ml), also provided by Dr. Gretchen Anderson. Then, the membrane was washed 3 times with TBS-T and incubated with 0.2 μg/mL HRP-conjugated goat anti-rabbit IgG for 1.5 h. They it was washed again and revealed with Pierce ECL Substrate and GE Healthcare ECL Reagent prepared according to the manufacturer’s instructions.
Both microbiological and physicochemical assays of the present study were carried out following security protocols normalized in the Laboratorio de Servicios Analíticos (LSA) at the Universidad Católica del Norte wich has the ISO/IEC 17025 accreditation [41].
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