Advanced Materials Research Vol. 825 (2013) pp 384-387 © (2013) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.825.384
Hydrogen sulphide production at alkaline, neutral and acid pH by a bacterial consortium isolated from Peruvian mine tailing and wetland. Juliet Mormontoy1,a and Jasmin E. Hurtado2,b 1,2
Lab. Biotecnologia Ambiental, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia, AP 4313, Lima-Peru. a
[email protected], b
[email protected]
Keywords: SRB, pH, mixed cultures, sulphide, pH
Abstract: The gol aim of this study is to optimize the ability to produce hydrogen sulphide (H2S) in pure and mixed cultures of sulfate reducing bacteria (SRB) at pH variations from 9 to 5. Hydrogen sulphide produced by SRB reacts with dissolved metals in water or tailings generating highly insoluble metal sulfides and therefore the selective immobilization of different metals. Three strains of SRB were isolated from Orcopampa mine tailings and from the Pantanos de Villa wetlands, both located in Peru. Cultures were identified by microscopy, cultural characteristics and biochemical tests as production of desulfoviridin and growth in different substrates. The production of H2S by pure and mixed cultures was evaluated at: acid pH (5), neutral pH (7) and alkaline pH (9). The mixed culture consisted of all three isolated species: Desulfobacter sp. from mine tailings and Desulfovibrio desulfuricans and Desulfovibrio sapovorans from wetland sludges. Pure cultures of these three strains grew and produced H2S at both neutral and alkaline pH. At low pH no pure culture was able to grow and no production of H2S was detected. A mixed culture formed by the three isolated SRB was the only culture that grew and produced sulphide at the three different pH tested in shorter time (24 hours). The improvement of bacterial activity can be based in the metabolic diversity of the mixed culture able to use lactate and acetate as a result of the joint activity of these species. Energy obtained from the substrate is thus used more efficiently. Introduction Sulphate reducing bacteria (SRB) are widely used in bioremediation processes, because of their capacity to produce H2S [1]. Sulphide produced by SRB reacts with metals dissolved in contaminated water or in mine tailings, precipitating and immobilizing them, thereby facilitating their subsequent treatment. [2]. Different studies have been made to optimize the ability to produce hydrogen sulfide in pure cultures [3,4] and in mixed cultures [5] with non isolated strains. Also have been isolated acidophilic SRB strains able to grow at pH lower than 5 [6,7]. The use of only one axenic culture in environmental processes, where control of parameters is limited might be difficult because changes on conditions could affect growth and development. On the other hand, the use of mixed cultures increases considerably the tolerance and resistance to changes [8]. The research aim of this study is to assess the survival and production of H2S under variations from pH 9 to pH 5 by SRB in pure and mixed defined cultures.
Methodology Samples The samples were collected in Orcopampa mine tailings (pH 8.5) and from the wetland Pantanos de Villa sludges (pH 7.3), both are located in Peru. All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 190.43.76.252-04/10/13,15:38:56)
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Isolation and characterization of SRB. One gram of each sample was incubated in vials containing 4.5 ml of Postgate B medium [9]. Growth was evidenced by the change to black color, due to formation of iron sulphide. Vials were maintained at 30 °C in anaerobic conditions. The isolation of pure cultures was carried out using solid Postgate B medium as described [15]. After obtaining pure strains, they were identified using biochemical tests according to the Bergeys manual [10]. Desulfoviridin was evaluated using the technique described by McNeil et al. [11]. Production of hydrogen sulphide a different pH. The mixed culture was prepared by inoculating 0.1 mL of each pure culture in vials containing 5 mL of liquid Postgate B medium. It was incubated at 30°C in anaerobic conditions, for five days. Pure cultures and mixed cultures were cultivated in quadruplicate at acid pH (5), neutral pH (7) and alkaline pH (9). Chemical and Biological assays SRB produce H2S from ferrous sulfate (which is part of the Postgate B medium), the decrease of iron in the solution is an indicrect indication of the production of iron sulphide and is directly related to the production of hydrogen sulphide. Tritation with potassium dichromate was used to quantify the amount of total iron [12]. Gram staining was used to monitor bacterial predominance. Statistical analysis All data was analyzed using the statistical program SPSS version 18 (IBM Corporation, United States). Analysis of variance (ANOVA) was used to evaluate the average iron consumption of each type of culture, pure and mixed. Significant ANOVA tests were further analyzed using the Scheffé test. A value of P
