A simplified material and energy balance approach for process development and scale-up of Coniothyrium minitans conidia production by solid-state cultivation in a packed-bed reactor.

Production of conidia of the biocontrol fungus Coniothyrium minitans by solid-state cultivation in a packed-bed reactor on an industrial scale is feasible. Spore yield and oxygen consumption rate of C. minitans during cultivation on oats and three inert solids (hemp, perlite, and bagasse) saturated with a liquid medium were determined in laboratory-scale experiments. The sensitivity of the fungus to reduced aw, and the water desorption isotherms of the four solid materials were also determined. C. minitans is very sensitive to reduced aw: 50% inhibition of respiration was found at aw 0.95, spore formation was completely inhibited at aw 0.97. A simplified mathematical model taking into account convective and evaporative cooling was used to simulate temperature and moisture gradients in the bed during cultivation. Adequate temperature control can be achieved with acceptable air flow rates for all four solid matrices. Moisture control is the limiting factor for cultivation in a packed bed. Oats cannot be used due to the shrinkage and aw reduction caused by evaporative cooling. Of the three inert supports tested, hemp provides the best spore yield and control of water activity, due to its high water uptake capacity. A spore yield of 9 x 10(14) conidia per m(3) packed bed can be achieved in 18 days, using hemp impregnated with a solution containing 100 g dm(-3) glucose and 20 g dm(-3) potato extract. Sufficient water is predicted to be available after 18 days, to allow a higher initial nutrient concentration, which may lead to higher spore yields.     

Temperature control in a continuously mixed bioreactor for solid-state fermentation

A continuously mixed, aseptic paddle mixer was used successfully for solid-state fermentation (SSF) with Aspergillus oryzae on whole wheat kernels. 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 improves heat transport to the bioreactor wall, which reduces 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 paddle mixer indicated that wall cooling becomes insufficient at the 2-m(3) scale for a rapidly growing fungus like Aspergillus oryzae. Consequently, evaporative cooling will remain important in large-scale mixed systems. Experiments showed that water addition will be necessary when evaporative cooling is applied in order to maintain a sufficiently high water activity of the solid substrate. Mixing is necessary to ensure homogeneous water addition in SSF. Automated process control might be achieved using the enthalpy balance. The enthalpy balance for the case of evaporative cooling in the paddle mixer was validated. This work shows that continuous mixing provides promising possibilities for simultaneous control of temperature and moisture content in solid-state fermentation on a large scale.     

Validation of a model for process development and scale-up of packed-bed solid-state bioreactors.

We have validated our previously described model for scale-up of packed-bed solid-state fermenters (Weber et al., 1999) with experiments in an adiabatic 15-dm(3) packed-bed reactor, using the fungi Coniothyrium minitans and Aspergillus oryzae. Effects of temperature on respiration, growth, and sporulation of the biocontrol fungus C. minitans on hemp impregnated with a liquid medium were determined in independent experiments, and the first two effects were translated into a kinetic model, which was incorporated in the material and energy balances of the packed-bed model. Predicted temperatures corresponded well with experimental results. As predicted, large amounts of water were lost due to evaporative cooling. With hemp as support no shrinkage was observed, and temperatures could be adequately controlled, both with C. minitans and A. oryzae. In experiments with grains, strong shrinkage of the grains was expected and observed. Nevertheless, cultivation of C. minitans on oats succeeded because this fungus did not form a tight hyphal network between the grains. However, cultivation of A. oryzae failed because shrinkage combined with the strong hyphal network formed by this fungus resulted in channeling, local overheating of the bed, and very inhomogeneous growth of the fungus. For cultivation of C. minitans on oats and for cultivation of A. oryzae on wheat and hemp, no kinetic models were available. Nevertheless, the enthalpy and water balances gave accurate temperature predictions when online measurements of oxygen consumption were used as input. The current model can be improved by incorporation of (1) gas-solids water and heat transfer kinetics to account for deviations from equilibrium observed with fast-growing fungi such as A. oryzae, and (2) the dynamic response of the fungus to changes in temperature, which were neglected in the isothermal kinetic experiments.     

Heat and water transfer in a rotating drum containing solid substrate particles

In previous work we reported on the simulation of mixing behavior of a slowly rotating drum for solid-state fermentation (SSF) using a discrete particle model. In this investigation the discrete particle model is extended with heat and moisture transfer. Heat transfer is implemented in the model via interparticle contacts and the interparticle heat transfer coefficient is determined experimentally. The model is shown to accurately predict heat transfer and resulting temperature gradients in a mixed wheat grain bed. In addition to heat transfer, the addition and subsequent distribution of water in the substrate bed is also studied. The water is added to the bed via spray nozzles to overcome desiccation of the bed during evaporative cooling. The development of moisture profiles in the bed during spraying and mixing are studied experimentally with a water-soluble fluorescent tracer. Two processes that affect the water distribution are considered in the model: the intraparticle absorption process, and the interparticle transfer of free water. It is found that optimum distribution can be achieved when the free water present at the surface of the grains is quickly distributed in the bed, for example, by fast mixing. Alternatively, a short spraying period, followed by a period of mixing without water addition, can be applied. The discrete particle model developed is used successfully to examine the influence of process operation on the moisture distribution (e.g., fill level and rotation rate). It is concluded that the extended discrete particle model can be used as a powerful predictive tool to derive operating strategies and criteria for design and scale-up for mixed SSF and other processes with granular media.     

Verticillium lecanii spore production in solid-state and liquid-state fermentations

Verticillium lecanii has been recognized as an entomopathogen with high potential in biological control of pests. Two types of cultivation methods, the solid-state fermentation (SSF) and the liquid-state fermentation (LSF), were examined for V. lecanii. In SSF, the substrate types including rice, rice bran, rice husk, and the mixtures of these components were tested. The results showed that both cooked rice with appropriate water addition and rice bran gave significantly higher spore production of 1.5 2 10⁹ spores/g substrate and 1.4 2 10⁹ spores/g substrate, respectively. In LSF, SMAY liquid medium was used as a base, and the effects of environmental conditions on the spore production of V. lecanii were investigated. From the time course study, on the 9th day the spore yield reached 1.2 2 10⁹ spores/ml of broth at 24v°C, 150 rpm for this strain. A series of medium volumes in the shaker-flask have been tested for the requirement of aeration. The largest surface aeration test, one tenth of the medium volume in the shaker-flask for cultivation, gave the highest spore count. The optimal pH value was tested and the initial pH 5 in the SMAY medium produced a high spore density. Finally, V. lecanii spores from SSF and LSF were different in size, shape, and size distribution; while mean spore length from SSF was 6.1 7m, and mean spore length from LSF was 5.0 7m.     

A new two-phase kinetic model of sporulation of Clonostachys rosea in a new solid-state fermentation reactor

A new two-phase kinetic model of sporulation of Clonostachys rosea in a new solid-state fermentation (SSF) reactor was proposed. The model including exponential and logistic models was applied to study the simultaneous effect of temperature, initial moisture content, medium thickness and surface porosity of the plastic membrane on C. rosea sporulation. The model fits experimental data very well and allows accurate predictions of spore production. The maximum spore production achieved 3.360 × 10¹⁰ (spores/gDM), about 10 times greater than that in traditional SSF reactor(data not shown). The new reactor can provide two times sporulation surface area. Moisture content can be adjusted by changing the surface porosity to meet the spore production. Two mixings carried out during fermentation makes medium loose and results in a mass of new sporulation surface area. Therefore, the new SSF reactor would have great potential for application in bulk spore production of fungal biocontrol agents.     

Mixing characteristics and startup of anaerobic downflow stationary fixed film (DSFF) reactors

Tests to determine the mixing characteristics of the anaerobic downflow stationary fixed film (DSFF) reactor during startup showed that mixing characteristics affected performance. Different mixing profiles were obtained by keeping the same flow distribution system and by varying the number of clay channels (1, 4, and 25) in the DSFF reactors (2-32 L). Results with a clean bed reactor indicated a plug flow pattern with a relatively large extent of dispersion. Recirculation dramatically improved the mixing and the residence time distribution (RTD) changed to that of the completely mixed type. Multiple-channel reactors exhibited a dead space of ca. 12% of the total volume, likely a result of a less than optimally designed flow distributor. A startup period of 90 days was necessary to achieve a maximum loading rate of between 10 and 15 kg COD/m(3) day, a volumetric methane production rate of up to 3 m(3) (STP)/m(3) day and a COD reduction efficiency of up to 90%. For the first 50 days of operation, the difference in achievable volumetric loading rate and volumetric methane production rate was only related to the surface-to-volume ratio of the reactors and was not affected by the number of channels present. After 90 days, the bacterial growth on the support material was sufficient to dramatically increase the amount of dead space in the reactors, especially in multiple-channel reactors (up to 55% of their volume). As a result, the performance of these reactors deteriorated and overloading characteristics were observed. Other results showed that biogas production alone was not sufficient to improve reactor mixing and that little or no shortcircuiting or channelling occurred. Furthermore, the nonmethanogenic bacterial activity in the liquid phase was not affected by the degree of mixing but acetoclastic methanogenic and hydrogenophilic methanogenic activity in the liquid phase were reduced as the fluid flow pattern in the reactor improved.     

Mathematical modeling as a tool to investigate the design and operation of the zymotis packed-bed bioreactor for solid-state fermentation

Zymotis bioreactors for solid-state fermentation (SSF) are packed-bed bioreactors with internal cooling plates. This design has potential to overcome the problem of heat removal, which is one of the main challenges in SSF. In ordinary packed-bed bioreactors, which lack internal plates, large axial temperature gradients arise, leading to poor microbial growth in the end of the bed near the air outlet. The Zymotis design is suitable for SSF processes in which the substrate bed must be maintained static, but little is known about how to design and operate Zymotis bioreactors. We use a two-dimensional heat transfer model, describing the growth of Aspergillus niger on a starchy substrate, to provide guidelines for the optimum design and operation of Zymotis bioreactors. As for ordinary packed-beds, the superficial velocity of the process air is a key variable. However, the Zymotis design introduces other important variables, namely, the spacing between the internal cooling plates and the temperature of the cooling water. High productivities can be achieved at large scale, but only if small spacings between the cooling plates are used, and if the cooling water temperature is varied during the fermentation in response to bed temperatures.     

Combined discrete particle and continuum model predicting solid-state fermentation in a drum fermentor

The development of mathematical models facilitates industrial (large-scale) application of solid-state fermentation (SSF). In this study, a two-phase model of a drum fermentor is developed that consists of a discrete particle model (solid phase) and a continuum model (gas phase). The continuum model describes the distribution of air in the bed injected via an aeration pipe. The discrete particle model describes the solid phase. In previous work, mixing during SSF was predicted with the discrete particle model, although mixing simulations were not carried out in the current work. Heat and mass transfer between the two phases and biomass growth were implemented in the two-phase model. Validation experiments were conducted in a 28-dm3 drum fermentor. In this fermentor, sufficient aeration was provided to control the temperatures near the optimum value for growth during the first 45-50 hours. Several simulations were also conducted for different fermentor scales. Forced aeration via a single pipe in the drum fermentors did not provide homogeneous cooling in the substrate bed. Due to large temperature gradients, biomass yield decreased severely with increasing size of the fermentor. Improvement of air distribution would be required to avoid the need for frequent mixing events, during which growth is hampered. From these results, it was concluded that the two-phase model developed is a powerful tool to investigate design and scale-up of aerated (mixed) SSF fermentors.     

Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters

The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low-strength soluble wastewaters using ethanol as a model substrate was investigated in laboratory-scale reactors at 30oC. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to12 g COD/L . d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGBS reactor for the anaerobic treatment of low-strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate-biomass contact and adequate expansion of the substrate-biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (V(up)) was greater than 2.5 m/h. Under such conditions, an extremely low apparent K(s) value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at V(up) lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory-scale reactors. A. more important restriction of the EGSB reactor was the sludge washout occurring at V(up) higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L. d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold-up, the operation should be limited to an organic loading rate of 7 g COD/L d. and to a liquid up-flow velocity between 2.5 and 5.5 m/h (c) 1994 John Wiley & Sons, Inc.     

Approach to designing rotating drum bioreactors for solid-state fermentation on the basis of dimensionless design factors

The development of large-scale solid-state fermentation (SSF) processes is hampered by the lack of simple tools for the design of SSF bioreactors. The use of semifundamental mathematical models to design and operate SSF bioreactors can be complex. In this work, dimensionless design factors are used to predict the effects of scale and of operational variables on the performance of rotating drum bioreactors. The dimensionless design factor (DDF) is a ratio of the rate of heat generation to the rate of heat removal at the time of peak heat production. It can be used to predict maximum temperatures reached within the substrate bed for given operational variables. Alternatively, given the maximum temperature that can be tolerated during the fermentation, it can be used to explore the combinations of operating variables that prevent that temperature from being exceeded. Comparison of the predictions of the DDF approach with literature data for operation of rotating drums suggests that the DDF is a useful tool. The DDF approach was used to explore the consequences of three scale-up strategies on the required air flow rates and maximum temperatures achieved in the substrate bed as the bioreactor size was increased on the basis of geometric similarity. The first of these strategies was to maintain the superficial flow rate of the process air through the drum constant. The second was to maintain the ratio of volumes of air per volume of bioreactor constant. The third strategy was to adjust the air flow rate with increase in scale in such a manner as to maintain constant the maximum temperature attained in the substrate bed during the fermentation.     

Operating characteristics of solid-state fermentation bioreactor with air pressure pulsation

The development of solid state fermentation (SSF) technology is very important to the production of cellulase and ultimately to the utilization of natural cellulose. However, inadequate dissipation of heat generated by biological activities has prevented solid state fermentation from large-scale applications. The paper deals with the development of a novel SSF bioreactor with air pressure pulsation. By developing a measurement and control system under Virtual Instrument (VI) concept, performance of SSF bioreactor with pressure pulsation was studied by cultivating Trichoderma koningii in solid medium made of wheat bran and corncob. The cooling effects of pressure pulsation on solid porous beds are discussed. Experimental results show that pressure pulsation enhances medium moisture evaporation, and hence, heat dissipation. Furthermore, through changing the pressure pulsation directions, it is able to mitigate the temperature gradients in the bioreactor. To sum up, pressure pulsation can provide the microbes with a growing environment at optimal temperature and medium water content.     

Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid-state fermentation system

Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation. This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF. In cultures of A. oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus. This is due to the fact that A. oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid. It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A. oryzae than was previously reported for R. oligosporus and C. minitans. It also means that the overall oxygen uptake rate for A. oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R. oligosporus and C. minitans. This would imply that cooling problems become more pronounced. Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF.     

Solid state fermentation reactors: from lab scale to pilot plant

Relatively many workers in the world are studying different aspects in SSF processes but few are working on reactor design and scale-up. From about 10 years, we are developing reactors from lab scale to pilot plant, based on the same technology, reactor design and flowsheet to allow fermentation with a deep layer (up to 1 m in the pilot plant). These reactors have all a forced aeration and the possibility or not to agitate. Regulations of temperature and water content of the culture are monitored by a special device.     

Preparation of fungal starter culture in liquid fluidized bed reactor

Summary Pregerminated Trichoderma reesei (Rut C-30) spores were grown on corncob particles in a liquid fluidized bed reactor (LFBR). Hyphal mass covered particles were recovered from the top of the reactor in 24 h and used as starters for solid substrate fermentation. The starter from LFBR gave better biomass production than spore or mycelial inoculum.     

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O₂, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.     

Deep-bed solid state fermentation of sweet sorghum stalk to ethanol by thermotolerant Issatchenkia orientalis IPE 100

A solid state fermentation (SSF) of sweet sorghum stalk to ethanol was conducted in 250-mL flask using thermotolerant Issatchenkia orientalis IPE 100, and the optimal operation parameters were determined as 42°C fermentation temperature, 75% (w/w) water content, 2mm particle size and 3% (w/w) inoculation rate in 250-mL conical flask. When the SSF was scaled up from the flask to a 10-L bioreactor, temperature gradient in the substrate bed was observed due to heat accumulation in the bioreactor. The temperature gradient was dependent on both substrate depth and operation temperature. Due to high thermotolerance of the strain IPE 100, a deep-bed SSF of sweet sorghum stalk was developed in the bioreactor. The highest ethanol yield of 0.25 g-ethanol/g-dry stalk was obtained at 37°C with 15-20 cm substrate depth in the bioreactor. These results provided a great potential for large-scale deep-bed SSF in practice.     

Modeling the liquid flow in up-flow anaerobic sludge blanket reactors

By means of stimulus-response experiments an Li(+) tracer, models for the fluid flow in a 30-m(3) UASB reactor, used for the anaerobic treatment of wastewater, were tested. From the model with the best fit it could be derived that both the sludge bed and the sludge blanket can be described as perfectly mixed tank reactors with short-circuiting flows; the settler volume acts like a plug-flow region.Apart from the volumes of the different flow regions, two parameters are necessary and sufficient to describe the fluid flow in a well functioning UASB reactor, i.e., the short-circuiting flow over the sludge bed and the short-circuiting flow over the sludge blanket. The volumes could be measured accurately.The short-circuiting flow over the sludge bed is a linear function of the sludge bed height. When the optimal height of the sludge bed is defined as the height for which the short-circuiting flows are as small as possible, a bed-height of 3.5-4 m is sufficient (for superficial gas velocities between 1 and 1.5 m/h). This is in contradiction to the results of other authors. The short-circuiting flows over the sludge bed and the sludge blanket were also influenced by the superficial gas velocity.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

Chemical States of Bacterial Spores: Heat Resistance and Its Kinetics at Intermediate Water Activity

Bacterial spore heat resistance at intermediate water activity, like aqueous and strictly dry heat resistance, is a property manipulatable by chemical pretreatments of the dormant mature spore. Heat resistances differ widely, and survival is prominently nonlogarithmic for both chemical forms of the spore. Log survival varies approximately as the cube of time for the resistant state of Bacillus stearothermophilus spores and as the square of time for the sensitive state. A method for measuring heat resistance at intermediate humidity was designed to provide direct and unequivocal control of water vapor concentration with quick equilibration, maintenance of known spore state, and dispersion of spores singly for valid survivor counting. Temperature characteristics such as z, E(a), and Q(10) cannot be determined in the usual sense (as a spore property) for spores encapsulated with a constant weight of water. Effect on spore survival of temperature induced changes of water activity in such systems is discussed.