Evaluation of quantitative real-time polymerase chain reaction for enumeration of biomining microorganisms in culture

The microbial populations of biomining communities are complex and diverse, including bacteria, archaea and fungi. Low pH, abundance of mineral particles, presence of microorganisms in biofilms and the species diversity of microorganisms inhabiting the biomining environment has made studying these populations difficult. Quantitative real-time polymerase chain reaction (Q-PCR) is increasingly used for the enumeration of microorganisms in the environment and has been used to study the populations in various biomining systems. This study evaluates the use of Q-PCR for the enumeration of biomining microorganisms in liquid cultures. Spectrophotometric quantification of extracted DNA was compared with a SYBR Green Q-PCR assay. The 16S rRNA gene was targeted for Q-PCR quantification of three biomining bacteria: Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, Sulfobacillus thermosulfidooxidans and the archaeon Ferroplasma acidiphilum. Primers were designed to amplify a 390 ± 35 bp region of the 16S rRNA gene. Southern blot hybridisation was used to determine the number of 16S rRNA gene copies per genome. Standard curves were constructed with DNA from Sinorhizobium meliloti and At. ferrooxidans. Using a standard curve constructed with At. ferrooxidans DNA there was no statistical difference between quantification using Q-PCR and the NanoDrop spectrophotometer (P < 0.05). However, there was a statistical difference when quantifying the archaeon (P > 0.05).