Development and application of quantitative real-time PCR based on the mitochondrial cytochrome oxidase subunit I gene for early detection of the grazer Poterioochromonas malhamensis contaminating Chlorella culture

Poterioochromonas malhamensis is a flagellate grazer that readily contaminates Chlorella cultures and can have a devastating impact on them, especially in large-scale culture. Here, we developed a quantitative real-time polymerase chain reaction (qPCR) method to detect and quantify P. malhamensis from high-density Chlorella cultures. A set of primers specific to the mitochondrial cytochrome oxidase subunit I gene (COI) of P. malhamensis was screened and characterized comprehensively for specificity, sensitivity and applicability to the field samples. The results showed that the primers were sufficiently specific to distinguish P. malhamensis from a wide variety of micro-eukaryotes which often coexist with P. malhamensis in microalgal cultures, and could detect a very low concentration of P. malhamensis from high concentrations of Chlorella sorokiniana (106, 107, and 108 C. sorokiniana cells mL−1). Moreover, the qPCR method was applied successfully to the monitoring of P. malhamensis population dynamics in Chlorella cultures in 100 L open ponds. Finally, the qPCR method was applied to the detection of this flagellate in the wider environment of the culture facility. It showed that the flagellate was widely distributed in the environment, and we speculate that Poterioochromonas in Chlorella cultures could originate from the air, the water, and the seed cultures. The qPCR method developed therefore proved to have the specificity and efficiency to be useful for early detection of P. malhamensis in industrial-scale Chlorella cultivation.