(567a) Development of a Quantitative Real-Time PCR Method to Measure Fungal Biomass for Determination of Growth Kinetics On Solid Media

Authors: 
Arquiza, J. A., Cornell University
Hunter, J. B., Cornell University


Fungi can colonize a wide variety of solid materials and their presence can be detrimental or beneficial, depending on the species involved. In either case, a reliable way of measuring biomass is needed in order to monitor the fungal growth rate on the substrate. Conventional methods reported in the literature include the following: measurement of colony diameter, evaluation of colony forming units (standard plate counts), direct weighing of biomass, visual measurement of hyphal length under a microscope, and the correlation of biochemical markers such as chitin, glucosamine or ergosterol to biomass. Among these, it is only the biochemical assays that can estimate fungal biomass growing in three dimensions, that is, with mycelial growth within the matrix of the solid substrate. Ergosterol measurements have been proven to be the most reliable but it cannot distinguish between different fungal species since the amount measured will be from all the fungi in the sample. In this study we investigate the use of the species-specific real-time polymerase chain reaction method (rt-PCR) to quantify the DNA of fungi in samples with the objective of using it to measure growth kinetic parameters. The development of an rt-PCR assay will allow the modeling of fungal growth with extensive substrate infiltration. The growth rates of individual species in the presence of other fungi can be also be measured. In this method, the ratio of the amount of DNA to biomass is a crucial parameter for converting the quantified DNA to biomass. The effect of the fungal species, age of colony, and water activity of media on this ratio was obtained. The fungi studied were the common food spoilage fungi Aspergillus niger, Penicillium chrysogenum and Eurotium amstelodami. The kinetic parameters of the growth of these fungi on food was measured using the developed rt-PCR assay. In the case of Aspergillus niger, the relationship between the production of extracellularly secreted amylase to the growth rate was determined. This would be useful in the design of enzyme production from fungi by solid-state fermentation.