A disadvantage of most microbial production processes is the need for sterile techniques. The objective of this study was the development of a robust fungal system allowing monoseptic growth with a minimum of sterile technique in plastic barrels. Selective growth conditions were achieved by mineral salts medium, known for the cultivation of Botrytis cinerea, but containing rapeseed oil instead of glucose as the sole source of carbon and energy. Furthermore, pH 3 was adjusted. A screening of fungi suitable for that system revealed Phialemonium curvatum AW02 isolated from compost. P. curvatum AW02 was superior in comparison with four further fungal isolates because high titers of hydrophilic spores were found in submerged production. Second, a biofilm formation on plastic segments or moving beds made harvesting of the biomass comfortable. Cultivations with volumes of 100 or 350 L showed no contaminations by bacteria when all conditions were controlled. Two independent approaches showed the dependance of growth on lipases in the cultivation system. A B. cinerea strain knocked out in lip1 showed reduced growth in comparison to the wild type because the first catabolic step is the triglyceride hydrolysis. P. curvatum AW02 lipase activity was detected. More than 90% was found to be cell wall associated. Solid shear stress liberated two active proteins showing IEPs of 4.7 or 5.6.

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A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5-1% tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1% tributyrin emulsion in 2% agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20-60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

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To set a benchmark in fungal growth rate, a differential analysis of prototrophic Aspergillus fumigatus AR04 with three ascomycetes applied in > 10 3 t year -1 scale was performed, i.e. Ashbya gosspyii (riboflavin), Aspergillus niger (citric acid) and Aspergillus oryzae (food-processing). While radial colony growth decreased 0.5-fold when A. gossypii was cultivated at 40°C instead of 28°C, A. fumigatus AR04 responded with 1.7-fold faster hyphal growth. A. niger and A. oryzae formed colonies at 40°C, but not at 43°C. Moreover, all A. fumigatus strains tested grew even at 49°C. In chemostat experiments, A. fumigatus AR04 reached steady state at a dilution rate of 0.7 h -1 at 40°C, 120% more than reported for A. gossypii at 28°C. To study mycelial growth rates under unlimited conditions, carbon dioxide increase rates were calculated from concentrations detected online in the exhaust of batch fermentations for 3 h only. All rates calculated suggest that A. fumigatus AR04 approximates Arrhenius' rule when comparing short cultivations at 30°C with those at 40°C. Linearization of the exponential phase and comparison of the slopes revealed an increase to 192% by the 10°C up-shift.