Quantification of biomining microorganisms using quantitative real-time polymerase chain reaction

Mineral bioleaching heaps represent an extreme environment: low pH, heat and high levels of metals are just some of the factors present in these systems. Despite this, there is a diverse range of microorganisms able to inhabit this environment. The inability to cultivate the majority of these organisms has severely hampered attempts to study the ecology of these environments as cultivation techniques reveal only a small fraction of the microbial diversity. Quantitative real-time polymerase chain reaction (Q-PCR) is a culture independent method that is increasingly being used for the enumeration of environmental microorganisms. A SYBRGreen Q-PCR assay was developed to quantify four biomining microorganisms in mixed cultures consisting of three bacteria: Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans and Sulfobacillus thermosulfidooxidans, and one archaeon: Ferroplasma acidiphilum. Enumeration of microorganisms by Q-PCR was tested against spectrophotometric quantification. When quantifying bacteria 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). In controlled systems, for example column leaching, where the microbial consortium of the environment is known, Q-PCR could be used to carry out high throughput, detailed population studies of the microorganisms within the system.