Toma de muestra

Muestra de suelo
Two composite soil samples will be collected from the top layer (010 cm) of each
location using sterilized plastic bags. One sample will be utilized for bacteria culture
and the other will be used to heavy metal analysis. The extractable heavy metal
concentrations in both soil samples will be measured by atomic absorption
spectrometry after extraction with aqua regia.

Muestra de agua

Cuantificacin de arsnico
Agua

Suelo

Aislamiento de cepas bacterianas

Each culture medium will be amended with arsenite (Na2HAsO37H2O) to give a final
concentrations of 0, 5, 50, 100, 150, 200, and 250mg As l -1 . If one bacteria resist this
concentrations, it will be utilized higher concentrations (500 and 750 mg As l -1)
(Takeuchi, 2007). For microbiological analysis, eight samples of each soil were
randomly collected and then mixed together to form two composite samples. Each
composite sample was put into plastic bags, transported to the laboratory where they
stored moist in the dark at 4C in polyethylene bags for until the analyses (Bachate
S.P. et al, 2009)

Evaluacin de la resistencia al As

Bacterial growth was monitored by measuring the optical density (OD) of the cultures
at 660 nm using a spectrophotometer (features). A single colony of each arsenic
resistant isolate shall be inoculated in triplicates into liquid CDM medium supplemented
with increasing concentrations (0, 5, 50, 100, 150, 200, and 250mg As l -1 ) of sodium
arsenite and shaken at 27 for one week. The MICs, defined as the lowest As (III)
concentration that completely inhibite the growth of each bacterium, were determined
(Lim and Cooksey, 1993).

Identificacin de las cepas

Par la identificacin de las bacterias se realizar la extraccin de DNA por medio de la
tcnica de CTAB, posteriormente se llevar a cabo la amplificacin del gen 16S rRNA
utilizando los oligonucletidos 27F 5AGAGTTTGATCMTGGCTCAG 3 y 1492R 5
GGTTACCTTGTTACGACTT 3`, el producto de PCR ser enviado a secuenciar a MCLAB
en San Francisco, CA, EEUU .Los secuencias obtenidas de la secuenciacin se
sometern a comparacin usando BLAST (NCBI) para determinar la posicin
taxonmica de las cepas bacterianas (Weisburg et al. 1991;Jha et al. 2011).

Bachate S.P. et al. (2009). Arsenic-resistant bacteria isolated from agricultural

soils of Bangladesh and characterization of arsenate-reducing strains. Journal of

Applied Microbiology, 145-156.

Takeuchi, e. a. (2007). Arsenic resistance and removal by marine and non-

marine bacteria. Journal of BIOTECHNOLOGY, 434-442.

Jha B, Gontia I, Hartmann A (2011) The roots of the halophyte Salicornia
brachiata are a source of new halotolerant diazotrophic bacteria with plant

growth-promoting
potential. Plant Soil. 356: 265-277.
Lim, C.K., and D.A. Cooksey. 1993. Characterization of chromosomal homologs
of the plasmid borne copper resistance operon of Pseudomonassyringae. J.

Bacteriol.175:44924498.
Weisburg W, Barns S, Pelletier D, David P, Gene L (1991) 16s Ribosomal DNA
Amplification for Phylogenetic Study. J Bacteriol.173 (2)697-703.
Pumpel, T., Pernfu_, B., Pigher, B., Diels, L., Schinner, F., 1995. A rapid screening
method for the isolation of metal-accumulating microorganisms. J. Ind. Microbiol.
14, 213217. (BUSCAR).