Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate.

Sabrina Hedrich,Axel Schippers,Mickaël Charron,Anne-Gwenaëlle Guézennec,Catherine Joulian

doi:10.3389/fmicb.2016.02044

Abstract

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

Highlights

Bioleaching, the extraction of metals by means of microorganisms, is nowadays a well-established process and an economic alternative to conventional roasting or pressure oxidation techniques for sulfidic low-grade ores
This study aims to develop and evaluate a selection of molecular methods to monitor the microbial community in bioleaching operations in order to define specific, quick, and reliable methods to be applied in further monitoring studies
The bioleaching culture used was the KCC consortium successfully applied in previous Cu concentrate bioleaching experiments containing Leptospirillum ferriphilum, Sulfobacillus benefaciens, Sulfobacillus thermosulfidooxidans, and Acidithiobacillus caldus (Spolaore et al, 2011)

Summary

Introduction

Bioleaching, the extraction of metals by means of microorganisms, is nowadays a well-established process and an economic alternative to conventional roasting or pressure oxidation techniques for sulfidic low-grade ores This environmental-friendly and low cost technology becomes especially important in the current context, where mineral resources are becoming more complex and Quantitative Monitoring of Bioleaching Species of lower grade. They comprise culture-dependent (plating, MPN counts) and biomolecular approaches such as the genetic fingerprinting techniques, terminal restriction fragment length polymorphism (T-RLFP; Wakeman et al, 2008), capillary electrophoresis single-strand conformation polymorphism (CE-SSCP; Foucher et al, 2003), denaturating gradient gel electrophoresis (DGGE; Demergasso et al, 2005; Halinen et al, 2009), microscopic methods like fluorescence in situ hybridization (FISH and CARD-FISH, Schippers et al, 2008), microarray approaches (e.g., Garrido et al, 2008; Remonsellez et al, 2009), quantitative real-time PCR (qPCR; Liu et al, 2006; Zhang et al, 2009), and generation sequencing techniques (e.g., Cardenas et al, 2016)

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Methods

Results