Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low‐grade copper sulfide ore monitored by real‐time PCR and oligonucleotide prokaryotic acidophile microarray

Abstract

SummaryThe bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium‐like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA‐based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium‐like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe3+/Fe2+ ion rate, are primary factors shaping the microbial dynamic in the heap.

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate

“…We tested qPCR assays reported in the literature to target Acidithiobacillus , Leptospirillum and Sulfobacillus species (e.g., Liu et al, 2006; Remonsellez et al, 2009; Zhang et al, 2009) and found that most of them were only specific on genus-level or not specific for the desired species (results not shown). Thus we designed new qPCR assays targeting the species of the KCC consortium.…”

“…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 next generation sequencing techniques (e.g., Cardenas et al, 2016). …”

“…While T-RFLP and CE-SSCP represent semi-quantitative methods based on PCR, qPCR is currently the most common method for quantitative microbial community monitoring. Even so qPCR is widely used and numerous assays have been described in the literature for the quantification of total bacteria, archaea, and special groups of microorganisms, there is only a limited number of assays published for bioleaching organisms (e.g., Liu et al, 2006; Remonsellez et al, 2009; Zhang et al, 2009). When searching for appropriate assays to monitor a defined bioleaching community it was found that most of these assays do not target the desired species or are not specific enough for quantification on species-level.…”

Environmental, Biogeographic, and Biochemical Patterns of Archaea of the Family Ferroplasmaceae

“…Detection or quantification of the Ferroplasmaceae in these environments was done mostly using small subunit (SSU) rRNA-targeting analyses, such as 16S rRNA gene clone libraries' sequencing, amplified rRNA gene restriction analysis (ARDRA) profiling, real-time quantitative PCR, restriction fragment length polymorphism (RFLP) or fluorescence in situ hybridization (FISH), and oligonucleotide microarray analysis, all of which revealed the presence of these archaea in a number of pyritic/arsenopyritic, gold-arsenopyritic/chalcopyritic, and lead-, zinc-, and copper-containing mines across all continents (11,24,26,45,46,51,55,56,57,58). Clones related to the family Ferroplasmaceae have also been documented in further natural sulfide-rich ecosystems, e.g., in the snottites, i.e., the stalactite-like formations of microbial origin taken from the walls of Frasassi Cave and in the Rio Garrafo cave systems, both located in Italy, where acidic microenvironments were formed as a result of sulfide oxidation (38).…”

A Theory-Based Pragmatism for Discovering and Classifying Newly Divergent Bacterial Species

“…The Species-Less model may apply to the case for pathogens, where immune-escape mutations may each constitute a new ecotype (Achtman and Wagner, 2008;Cohan, 2010). Also, the Species-Less model may apply in cases where an environment undergoes a succession process, where organisms at a site must adapt to rapidly changing conditions, for example the successions that occur on mine tailings, with pH and oxidation levels changing predictably and quickly (Remonsellez et al, 2009). …”