Aspergillus flavus produces dangerous secondary metabolites known as aflatoxins, which are toxic and carcinogenic, and their contamination of agricultural products results in health issues and economic hardships in the U.S. and around the world. Early identification of aflatoxigenic isolates of A. flavus is the key in the management of these fungi. An emerging detection method for specific fungi identification involves the analysis of microbial volatile organic compounds (MVOCs) released by the fungi. Complicating this approach is the understanding that many factors influence metabolic production, including growth parameters, such as growth media, temperature, spore counts and oxidation stress. In addition, analytical and data analysis methods can also influence the results. Several growth and analysis methods were evaluated and optimized in order to better understand the effect of the methods on fungi MVOC signatures. The results indicate that carboxen/polydimethylsiloxane (CAR/PDMS) has the best extraction efficiency for the MVOCs emitted by A. flavus. Both chemical defined agar (CDA) and chemical defined liquid (CDL) are suitable growth media for MVOC emission studies. The highest MVOC production was found at 30˝C. Log transformation was considered one of the best data pretreatment methods when analyzing MVOC data and resulted in the best principal component analysis (PCA) clustering in the experiments with different growth media. This study aims to elucidate fungal volatile organic compounds (VOCs) differences due to variations in growth parameters as a first step in the development of an analytical method for the monitoring of aflatoxigenic A. flavus contamination in crop storage facilities.

Fungi produce a variety of microbial volatile organic compounds (MVOCs) during primary and secondary metabolism. The fungus, Aspergillus flavus, is a human, animal and plant pathogen which produces aflatoxin, one of the most carcinogenic substances known. In this study, MVOCs were analyzed using solid phase microextraction (SPME) combined with GCMS from two genetically different A. flavus strains, an aflatoxigenic strain, NRRL 3357, and a non-aflatoxigenic strain, NRRL 21882. A PDMS/CAR SPME fiber was used over 30 days to observe variations in MVOCs over time. The relative percentage of individual chemicals in several chemical classes (alcohols, aldehydes, esters, furans, hydrocarbons, ketones, and organic acids) was shown to change considerably during the varied fungal growth stages. This changing chemical profile reduces the likelihood of finding a single chemical that can be used consistently as a biomarker for fungal strain identification. In our study, discriminant analysis techniques were successfully conducted using all identified and quantified MVOCs enabling discrimination of the two A. flavus strains over the entire 30-day period. This study underscores the potential of using SPME GCMS coupled with multivariate analysis for fungi strain identification.