Solid and liquid media for isolating and cultivating acidophilic and acid-tolerant sulfate-reducing bacteria

Abstract

Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro The main features of the 'standard' solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors.

Design and Application of a Low pH Upflow Biofilm Sulfidogenic Bioreactor for Recovering Transition Metals From Synthetic Waste Water at a Brazilian Copper Mine

“…The dominant dissolved transition metal present was copper, and it also contained smaller concentrations of cobalt, nickel, and zinc, but due to its relatively high pH, iron concentrations were very low (∼2 μM). Basal salts (Ñancucheo et al, 2016) were added to the synthetic mine water, together with glycerol (5 mM, as the principal electron donor) and 0.1 g yeast extract L -1 (to supply growth factors) and its pH adjusted to 5.0. The chemical composition of the amended synthetic mine water, and comparison with AMD at the Sossego mine, is shown in Table 1 .…”

Synthesis of Copper Sulfide Nanoparticles Using Biogenic H2S Produced by a Low-pH Sulfidogenic Bioreactor

“…However, most of the previously described SRB grow optimally at pH values between 6 and 8 [5]. Acidophilic SRB have been difficult to isolate and cultivate in vitro due to the challenges associated with enrichment and cultivation [7]. Two Desulfosporosinus spp.…”

“…The system was maintained at pH 4.5, 30 • C and operated at a 50 rpm stirring speed. The bioreactor was initially operated over a pre-trial period with SRB medium at pH 2.5 [7], containing 5 mM glycerol as electron donor and 0.01% (w/v) yeast extract. Automated flow into the sulfidogenic reactor was controlled by a pump linked to the pH unit of the control module.…”

“…Acetic acid was measured by HPLC coupled with a UV detector (Agilent Technologies, Santa Clara, CA, USA) [14], and glycerol was determined enzymatically using a commercial kit [15]. Bacteria were isolated from the bioreactor by streaking liquid samples onto overlay SRB medium to promote the growth of acidophilic SRB [7]. Phylogenetic analysis of the isolates was carried out using the method described by Rowe and colleagues [16].…”

“…Attempts to cultivate acidophilic or acid-tolerant SRB from environmental samples have been mostly unsuccessful; nevertheless, media formulations have proven successful when enriching mixed populations possessing acid-tolerant or acidophilic sulfate-reducing properties, e.g., a microbial mat collected from an abandoned copper mine in Spain [4]. This enrichment process has also been used to set up anaerobic bioreactors for the remediation of AMD and to selectively precipitate transition metals as sulfides [4,6,7].…”