# Effects of Growth Parameters on the Analysis of _Aspergillus flavus_ Volatile Metabolites
## 1 Introduction  
Aflatoxins are secondary metabolic products produced primarily by _Aspergillus flavus_ and _Aspergillus parasitic_[1]. Aflatoxin contamination of corn, peanut and other agricultural commodities has a significant impact on health and the agricultural economy, especially in the southeastern United States, where aflatoxin contamination costs farmers, buyers and sellers an annual average of $22.7 million [2]. The production of aflat is always associated with the production of other metabolites, some of which are volatile. These volatile metabolites are produced during both primary and secondary metabolism and are often collectively referred to as microbial volatile organic compounds (MVOCs) [3]. MVOCs are widely investigated as the indicator of fungal growth [4; 5; 6; 7], mycotoxins' production [8; 9; 10] and for fungal taxonomy [11; 12; 13]. In recent years, metabolomics approaches have been widely used for the investigation of the metabolites of biological samples for identifying biomarkers that correlate to a disease [14; 15], drug toxicity [16; 17] or genetic or environmental variation [18]. Metabolites can belong to a wide variety of compound classes, such as amino acids, lipids, organic acids, nucleotides, alcohols, esters and hydrocarbons [19]. These compounds are very diverse in their physical and chemical properties and occur in a wide concentration range. Some of these metabolites are volatile enough for headspace sampling. Metabolic profiling and fingerprinting methods are used to elucidate a microorganism's life processes [19]. Metabolic profiling is a determination of the chemicals and their concentrations produced by the specific biosynthesis pathway of organisms. Metabolic fingerprinting is the screening approach to classify samples based on metabolite patterns or \"fingerprints\". The metabolomics study process often includes sample preparation, sample collection, instrumental analysis, data pretreatment and data analysis. Relative humidity, temperature, substrate (growth medium), number of fungal spores inoculated and oxidation stress are the main factors influencing fungal growth, metabolism and MVOCs' production in a laboratory setting [20; 21; 22]. For example, [PERSON] _et al._[23] studied the effect of incubation temperature (10-30 \({}^{\circ}\)C), pH (3.0-4.0) and vanillin concentration (350-1200 ppm) on the growth of _A. flavus_. They concluded that the germination time and radial growth rate were significantly affected by the three studied variables. [PERSON] and [PERSON] [24] studied the effects of light, temperature and pH value for aflat' production. They indicated that 24 \({}^{\circ}\)C was the optimal temperature for aflatoxin production. [PERSON] _et al._[21] studied the influence of various growth parameters on fungal growth and volatile metabolite production by indoor molds. They proved that the range of MVOCs and the quantities were larger on malt extract agar than on wallpaper and plasterboard. VOCs and aflatoxin production have also been shown to be associated with oxidative stress [25]. Clearly, fungal growth conditions are an important consideration when conducting a metabolomic fingerprinting study involving the production of MVOCs. Sampling methods, such as thermal desorption tube (Tenax TA) [4; 26; 27], purge and trapping of headspace gases [28; 29], headspace sorptive extraction [12; 30] and solid phase microextraction (SPME) [31; 32; 33; 34], have been used for the collection of MVOCs. SPME is a popular technique because it has the advantages of low cost per analysis and portability. Volatile chemicals can be selectively enriched on SPME fibers depending on fiber coating selection. Therefore, the SPME fiber coating selection is important and should be tailored for specific applications. Optimization of fiber selection for 15 volatile and semivolatile analytes representing 13 organic classes was performed, and extraction efficiencies of the fibers for each of the analytes were compared [35].