The Production of Hydrocarbons and other Lipids by Trichoderma koningii, Penicillium janthinellum and their Mixed Species Culture Grown Aerobically on Four Different Carbon Substrates

The fungal species, Trichoderma koningii, Penicillium janthinellum, and a mixture of Trichoderma koningii and Penicillium janthinellum were tested to determine their abilities to convert different carbon substrates (undecanoic acid (UDA), decanoic acid (DA), mixture of decanoic and undecanoic acid (UDA+DA), and mixture of potato dextrose broth and undecanoic acid (UDA+PDB) into different cellular metabolites. The mixed species culture showed higher biomass and carbon dioxide production on all 4 substrates compared to individual cultures. The mixed species culture showed highest biomass production of 59.5 mg/ml and cumulative carbon dioxide production of 394.2 mg after 576 hours of growth, when grown on mixture of potato dextrose broth and undecanoic acid. The intracellular accumulation of lipids in mixed species culture and single cultures was confirmed by fluorescent microscopy during stationary and exponential phase when UDA+PDB is source of carbon.;The analysis of fungal culture samples by pyrolysis-field ionization mass spectrometry (Py-FIMS) supported analysis by TLC-FID, and showed that fatty acid concentrations ranged between 4.4 and 6.6% of total molecular ion intensities (TIIs) depending on carbon substrate and ranged from n-C11 to n-C18. The Py-FIMS analysis shows that the intracellular hydrocarbons including n- alkanes, alkenes and n-alkyl esters were most abundant components with total yield ranging between 7.4 and 15.7% of total ion intensities (TIIs). When fungal cells were grown on fatty acids and PDB the C-chain lengths of extracellular alkanes ranged from n-C19 to n-C34, alkenes n-C30:1- nC40:1, and long chain alkyl esters from C41 - C60:1. According to Py-FIMS analyses, alkanes, alkenes and alkyl esters were not observed in a chloroform extract of PDB. The latter indicates that the studied mixed fungal species catalyze the conversion of fatty acids to hydrocarbons during growth.;Chemical analysis of different lipids produced by mixed species culture was analyzed by Iatroscan TLC-FID to identify 15 different lipid classes produced. The highest total intracellular lipid content of 3.5 mg/ml was observed when mixed species culture was grown on PDB+UDA during the exponential growth phase. Free fatty acids were the most dominant lipid class produced intracellularly and extracellularly during both the exponential and stationary growth phases. They account for >80% of total lipids produced. Analysis of intracellular and extracellular saturated and unsaturated fatty acids by GC-FID analysis shows, out of 55 fatty acids analyzed, the most dominating fatty acids on all substrates were palmitic acid (C16), stearic acid (C18 ), oleic acid (C18:1), and linoleic acid (C18:2), which account for nearly 70% of total free fatty acids (FFA) produced. The concentration of monounsaturated fatty acids and polyunsaturated fatty acids were similar, and their content ranged from 23% to 66% of the total FFA (0.007 to 0.57 mg/ml), and concentration of saturated fatty acids ranged between 33% and 75% of the total FFA (0.01 to 1.97 mg/ml) depending on substrate type and growth phase.