Process control of solid-state fermentation : simultaneous control of temperature and moisture content

Solid-state fermentation (SSF), <em>i.e.</em> cultivation of micro-organisms on moist solid substrates in the absence of free-flowing water, is an alternative for submerged fermentation (SmF) for the production of biotechnological products. In recent years, research on SSF has led to a wide range of applications on lab scale, and comparative studies between SmF and SSF claim higher yields and other advantages for products made by SSF. In spite of these examples the commercial application of SSF processes in Western countries remains unusual mainly due to problems associated with scale-up. This thesis aims at solving one the most important scale-up problems, which is the simultaneous control of temperature and moisture content in a large-scale bioreactor.The use of mixed bioreactors for SSF was considered the first step in solving scale-up problems with respect to temperature and moisture-content control. Continuous mixing improved temperature control and prevented inhomogeneities in the bed. Respiration rates found in this system were comparable to those in small, isothermal, unmixed beds, which showed that continuous mixing did not cause serious damage to the fungus or the wheat kernels. Continuous mixing improved heat transport to the bioreactor wall, which reduced the need for evaporative cooling and thus may help to prevent the desiccation problems that hamper large-scale SSF. However, scale-up calculations for the mixed bioreactor indicated that wall cooling will become insufficient at a scale of 2 m <sup>3</SUP>for a rapidly growing fungus like <em>A. oryzae</em> . Consequently, evaporative cooling will remain important in large-scale mixed systems. Experiments showed that water addition is necessary when evaporative cooling is applied, to maintain a sufficiently high water activity of the solid substrate.Evaporative cooling is very important for large-scale bioreactors. However, it seriously dehydrates the solid substrate and will limit fungal growth. Water has to be added during fermentation to control the moisture content. A model is described, which estimates the extracellular (nonfungal) and overall water contents of wheat grains during solid-state fermentation (SSF). Model parameters were determined using an experimental membrane-based model system, which mimicked the growth of <em>A. oryzae</em> on the wheat grains and permitted direct measurement of the fungal biomass dry weight and wet weight. The model can be used to calculate the water addition that is required to control the extracellular water content in a mixed solid-state bioreactor.A control strategy is presented for simultaneous control of the temperature and moisture content during SSF in a continuously mixed paddle bioreactor. Evaporative cooling with varying air flow rate was applied to control the temperature of the solid substrate. The extracellular water content was controlled by adding a fine mist of water droplets onto the mixed solid substrate, using the water balance model to calculate the required addition based on on-line measurements. Temperature and extracellular water content were successfully controlled, which resulted in an improved biomass production compared to similar fermentations with temperature control only. No negative effects of water addition were observed with regard to biomass production. Control aimed at constant extracellular water content was shown to be superior to control aimed at constant overall water content of the fermented solids.The use of <sup>1</SUP>H-NMR imaging is described as a powerful technique to study solid-state fermentation at particle level. Gradients inside substrate particles cannot be prevented in solid-state fermentation. We report gradients in moisture and glucose content during cultivation of <em>A. oryzae</em> on membrane-covered wheat-dough slices, which were calculated from <sup>1</SUP>H-NMR images measured <em>in vivo</em> . We found that moisture gradients in the solid substrate remain small when evaporation is minimized. This is corroborated by predictions of a diffusion model. In contrast, strong glucose gradients developed. Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels. Integration of the glucose profile gave an average concentration close to the measured average content. Based on published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity. The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this non-conventional fermentation environment.Finally, an overview is given of recent advances in process control in large-scale SSF systems. In addition, two commercially available bioreactors are discussed with respect to process control: the koji bioreactor and alternative bioreactors based on industrial solid mixers, which both can facilitate the scale-up of SSF.