Mimicking gas and temperature changes during enzyme production by Rhizopus oligosporus in solid-state fermentation Lilik Ikasari and David A. Mitchell* Department of Chemical Engineering, The University of Queensland, Brisbane, Queensland 4072, Australia. Fax +61 7 33654199. E-mail davidmi@cheque.uq.edu.au Step changes in the gas environment and temperature during membrane culture of Rhizopus oligosporus were made to mimic those changes which arise during solid-state fermentation due to mass and heat transfer limitations. A decrease of O 2 concentration from 21% to 0.5% did not alter protease production by R. oligosporus but retarded amyloglucosidase production. An upshift from 37°C to 50°C decreased the activities of both enzymes, with the effect on protease being due to enzyme deactivation rather than a decrease in production. Introduction Fungal growth and product formation in solid-state fer- mentation (SSF) are affected by environmental conditions, such as temperature, pH, and O 2 and nutrient concentra- tions (Brown, 1988). However, fungal activity also changes the local environment due to nutrient and O 2 consump- tion, metabolite production and metabolic heat release. Gas concentration and temperature gradients commonly develop due to the mass and heat transfer limitations in SSF (Barstow et al., 1988; Ghildyal et al., 1992; 1993). Oxygen levels in SSF systems typically start at optimal levels, but then decrease to quite low values due to transfer limitations, especially when the moisture content within the substrate bed is high, or due to rapid respiration when high biomass density is attained. However, the effect of the gas environment is typically studied by applying a range of different gas phases to different cultures, with each culture being exposed to the particular gas phase from the start of the fermentation. In addition, although O 2 and CO 2 are usually exchanged during respiration in SSF, in experi- mental studies the O 2 concentration is typically varied while keeping the CO 2 concentration constant, or vice versa. Metabolic heat is not easily dissipated from the system, due to the poor heat transfer properties of solid substrates (Mudgett, 1986). The resulting overheating decreases enzyme production rates, extends production periods and deactivates enzymes (Brown, 1988; Ghildyal et al., 1993). Typically the effects of temperature on enzyme production are investigated by incubating a number of cultures at a range of temperatures which are maintained throughout the growth cycle (the ‘‘isothermal approach’’). However, in SSF the process starts at the optimal temperature and the temperature increases some time later. The effect of changes in the environment during SSF will differ from the effect of low O 2 concentration or high temperature applied from the start of the fermentation. In this study the gas environment and incubation tempera- ture were changed during enzyme production by Rhizopus oligosporus to mimic changes typical of SSF. Since RQ values are typically around 1 in SSF (Okazaki et al., 1980), the sum of the O 2 and CO 2 concentrations was maintained at 21% (v/v). However, since this was an initial investigation the gas concentration and temperature changes were made as step changes rather than gradual changes. Membrane culture enabled biomass to be measured directly, allowing calculation of specific enzyme production. Materials and methods Membrane culture method Rice bran model substrate contained 15 g rice bran, 4 g - carrageenan and 100 ml water, which were mixed, boiled and poured into plastic dishes of 4 cm diameter and 5 mm depth to form slabs. Once the gel set, the slab was overlaid with a wet 47 mm diameter polycarbonate membrane filter of 0.2 m pore size (Poretics No.13013). A spore suspen- sion (50 l) prepared from Rhizopus oligosporus ACM 145F (= ATCC 96528) containing approximately 10 7 spores/ml was spread across the membrane filter surface. At each sampling time, the mycelial mat was peeled, weighed and used for crude enzyme preparation. A separate experiment Biotechnology Letters, Vol 20, No 4, April 1998, pp. 349–353 © 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 4 ⋅ 1998 349