# Effects of Growth Parameters on the Analysis of _Aspergillus flavus_ Volatile Metabolites
## 3 Results and Discussion  
### Evaluation of SPME Fiber on Metabolic Profiling  
Extracted MVOC profiles and quantities were determined, and the information was used to select the best SPME fiber for metabolic fingerprinting. Experiment precision (repeatability) was evaluated based on the Relative Standard Deviation (RSD%) of the six replicates of three SPME fiber types: CAR/PDMS, DVB/PDMS and DVB/CAR/PDMS. These fibers were evaluated in terms of their efficiency in extracting volatile metabolites emitted by _A. flavus_ K73 growth on an MEA substrate. The fungal culture was incubated for 7 days at 30 \({}^{\circ}\)C with an initial inoculation spore concentration of \(1\times 10^{6}\) spores/mL. The SPME extraction was maintained at 30 \({}^{\circ}\)C for 5 h. The extraction efficiency evaluation included two aspects, MVOC selectivity and quantity (peak area). Three evaluated fibers showed different abilities to extract volatile metabolites of _A. flavus_, as shown in Figure 1A,B. We took steps to reduce the impact of this natural variation for collecting Figure 1 data. The 18 samples (six replicates for each fiber) that were analyzed were grown under identical conditions at the same time. The large error bars indicate the degree of natural variation. However, even with the large error bars, we are able to determine that CAR/PDMS is superior at extracting VOC, resulting in a larger total ion count. The CAR/PDMS fiber not only extracted the largest number of MVOCs (Figure 1A), but also extracted the largest amount of MVOCs based on the total peak area of all of the volatile metabolites (Figure 1B). A closer look at the data revealed MVOC functional group selectivity. Identified MVOCs were divided into nine chemical classes, including alcohols, aldehydes, furans, hydrocarbons, ketones, organic acids, organosulfur compounds, sesquiterpenes and other compounds. Among the chemical classes, hydrocarbons were divided into Hydrocarbon 1 (fewer than ten carbons) and Hydrocarbon 2 (ten or more carbons). Other compounds include seven unknown compounds, one ether and one ester. The CAR/PDMS fiber extracted greater amounts of alcohols, furans, Hydrocarbon 1, Hydrocarbon 2 and ketones, while DVB/PDMS extracted larger amounts of high molecular weight compounds containing the organosulfur compounds, sesquiterpenes and other compounds (Figure 2). These results agree with the literature, which describes the CAR/PDMS as likely to extract low molecular weight compounds, while DVB/PDMS is better at extracting high molecular weight compounds [56]. Figure 1: Solid phase microextraction (SPME) fibers’ comparison through the number of (**A**) and amount of (**B**) volatile metabolites extracted from _A. flavus_ culture using three types of SPME fibers, carboxen (CAR)/PDMS, divinylbenzene (DVB)/CAR/PDMS and DVB/PDMS, in six replications. The error bars indicate the standard deviation of six replicates. Since the CAR/PDMS fiber has difficulty adsorbing higher molecular weight analytes and DVB/PDMS has difficulty extracting analytes with low molecular weights, a DVB/CAR/PDMS fiber was developed by the manufacturer [57]. The advertised extraction advantage of the extended molecular weight range of VOCs was not observed based on our results (Figure 1A,B). CAR/PDMS was determined to be an excellent SPME fiber coating choice for fungal MVOC profiling based on its ability to collect the largest number and greatest quantity of MVOCs. The precision or repeatability of this method was examined using the relative standard deviation percentage (RSD%) of each extracted and identified fungal MVOC. The RSD% of each metabolite was calculated using both peak area and peak area percentage data. An internal standard (IS) was not used in this study for two reasons: (1) concerns that an internal standard would compete with absorption sites on the SPME fibers with fungal VOCs; and (2) the ultimate goal of this program is the development of an electronic nose for early detection of _A.