Substrate aggregation due to aerial hyphae during discontinuously mixed solid-state fermentation with Aspergillus oryzae: experiments and modeling

Solid-state fermentation (SSF) is prone to process failure due to channeling caused by evaporative cooling and the formation of an interparticle mycelium network. Mixing is needed to break the mycelium network and to avoid such failure. This study presents the first attempt to quantify and predict the effect of mycelium bonds on particle mixing and vice versa. We developed a novel experimental set-up to measure the tensile strength of hyphal bonds in SSF: Aspergillus oryzae was cultivated between two wheat-dough disks and the tensile strength of the aerial mycelium was measured with a texture analyzer. Tensile strength at different incubation times was related to oxygen consumption, to allow a translation to a rotating drum with A. oryzae cultivated on wheat grain. We performed several discontinuously mixed solid-state fermentations in the drum fermentor and measured the number and size of grain-aggregates remaining after the first mixing action. We integrated data on mycelium tensile strength into a previously developed two-dimensional discrete-particle model that calculates forces acting on individual substrate particles and the resulting radial-particle movements. The discrete-particle model predicted the quantity and size of the aggregates remaining after mixing successfully. The results show that the first mixing event in SSF with A. oryzae is needed to break mycelium to avoid aggregate formation in the grain bed, and not to distribute water added to compensate for evaporation losses, or smooth out temperature gradients.     

Model for on-line moisture-content control during solid-state fermentation

In this study we describe a model that estimates the extracellular (nonfungal) and overall water contents of wheat grains during solid-state fermentation (SSF) with Aspergillus oryzae, using on-line measurements of oxygen, carbon dioxide, and water vapor in the gas phase. The model uses elemental balances to predict substrate dry matter losses from carbon dioxide measurements, and metabolic water production, water used in starch hydrolysis, and water incorporated in new biomass from oxygen measurements. Water losses caused by evaporation were calculated from water vapor measurements. Model parameters were determined using an experimental membrane-based model system, which mimicked the growth of A. oryzae on the wheat grains and permitted direct measurement of the fungal biomass dry weight and wet weight. The measured water content of the biomass depended heavily on the moisture content of the solid substrate and was significantly lower than the estimated values reported in the literature. The model accurately predicted the measured overall water content of fermenting solid substrate during fermentations performed in a 1.5-L scraped drum reactor and in a 35-L horizontal paddle mixer, and is therefore considered validated. The model can be used to calculate the water addition required to control the extracellular water content in a mixed solid-state bioreactor for cultivation of A. oryzae on wheat.     

Aspergillus oryzae in solid-state and submerged fermentations. Progress report on a multi-disciplinary project

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications.     

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.     

Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.     

Solid-state fermentation in rotating drum bioreactors: operating variables affect performance through their effects on transport phenomena

Aspergillus oryzae ACM 4996 was grown on an artificial gel-based substrate and on steamed wheat bran during solid-state fermentations in 18.7 L rotating drum bioreactors. For gel fermentations fungal growth decreased as rotational speed increased, presumably due to increased shear. For wheat bran fermentations fungal growth improved under agitated compared to static culture conditions, due to superior heat and mass transfer. We conclude that the effects of operational variables on the performance of SSF bioreactors are mediated by their effects on transport phenomena such as mixing, shear, heat transfer, and mass transfer within the substrate bed. In addition, the substrate characteristics affect the need for and the rates of these transport processes. Different transport phenomena may be rate limiting with different substrates. This work improves understanding of the effects of bioreactor operation on SSF performance.     

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.     

Model-based bioreactor selection for large-scale solid-state cultivation of Coniothyrium minitans spores on oats

Non-mixed and mixed SSF reactors were evaluated for their applicability in large-scale spore production of the biocontrol fungus Coniothyrium minitans. The major problem to overcome in large-scale SSF is heat accumulation. Testing various cooling strategies in large-scale bioreactors would be very expensive and time consuming, therefore lab experiments in combination with mathematical simulations were used instead. The metabolic heat production rate, estimated from the oxygen consumption rate of C. minitans on oats in Erlenmeyer flasks, was about 500 Watt per m(3) bed. Conductive cooling in packed-bed reactors was insufficient to cool large reactor volumes (radius > 0.2 m). The poor thermal conductivity of the bed (lambda(b) = 0.1 W m(-2) K(-1)) resulted in steep radial temperature profiles. Adequate temperature control could be achieved with forced aeration, but concomitant water losses lead to significant shrinkage of the oats (30%) and critically low water activities, even though the bed was assumed to be aerated with water saturated air. Mixed systems, however, allowed heat removal without the need of evaporative cooling. Simulations showed that large volumes could be cooled via the wall at low mixing intensities and small temperature driving forces. Experimental studies showed no detrimental effect of mixing on spore production by C. minitans. The spore production yield in a continuously mixed scraped-drum reactor (0.2 rpm) was 5 x 10(12) spores per kg dry oats after 450 hours. Based on the scale-up potential of the mixed system and the absence of detrimental mixing effects it is believed that a mixed bioreactor is superior to a non-mixed system for large-scale production of C. minitans spores.     

Limitations of membrane cultures as a model solid-state fermentation system

AIMS: To examine the reliability of membrane cultures as a model solid-state fermentation (SSF) system. METHODS AND RESULTS: In overcultures of Aspergillus oryzae on sterilized wheat flour discs overlaid with a polycarbonate membrane, we demonstrated that the presence of membrane filters reduced the maximum respiration rate (up to 50%), and biomass and alpha-amylase production. We also show that the advantage of membrane cultures, i.e. total recovery of biomass, is not very evident for the system used, while the changes in metabolism and kinetics are serious drawbacks. CONCLUSIONS: The use of membrane cultures is artificial and without substantial benefits and therefore has to be carefully considered. SIGNIFICANCE AND IMPACT OF THE STUDY: In future studies on kinetics and stoichiometry of SSF, one should not completely rely on experiments using membrane cultures as a model SSF system.     

Improvement of the glaB promoter expressed in solid-state fermentation (SSF) of Aspergillus oryzae

The glucoamylase-encoding gene (glaB) promoter should be very useful for recombinant protein production in solid-state fermentation (SSF) of Aspergillus oryzae. A 97-bp fragment containing the cis-element of the glaB promoter was inserted into the glaA promoter, which was little expressed in SSF. The chimeric promoter showed about a 24-fold increase in promoter activity in SSF. Eight copies of the 97-bp fragment were tandemly fused with the glaB promoter. The improved promoter showed about a 4.6-fold increase in promoter activity in SSF. The glaB gene was overexpressed under control of the improved glaB promoter in SSF. Recombinant glucoamylase production reached about 1524 mg/kg-broth for 2 d. The improved glaB promoter should be very useful for overproduction of a recombinant protein in SSF of A. oryzae.     

A wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acid

In this study, a novel generic feedstock production strategy based on solid-state fermentation (SSF) has been developed and applied to the fermentative production of succinic acid. Wheat was fractionated into bran, gluten and gluten-free flour by milling and gluten extraction processes. The bran, which would normally be a waste product of the wheat milling industry, was used to produce glucoamylase and protease enzymes via SSF using Aspergillus awamori and Aspergillus oryzae, respectively. The resulting solutions were separately utilised for the hydrolysis of gluten-free flour and gluten to generate a glucose-rich stream of over 140gl(-1) glucose and a nitrogen-rich stream of more than 3.5gl(-1) free amino nitrogen. A microbial feedstock consisting of these two streams contained all the essential nutrients required for succinic acid fermentations using Actinobacillus succinogenes. In a fermentation using only the combined hydrolysate streams, around 22gl(-1) succinic acid was produced. The addition of MgCO(3) into the wheat-derived medium improved the succinic acid production further to more than 64gl(-1). These results demonstrate the SSF-based strategy is a successful approach for the production of a generic feedstock from wheat, and that this feedstock can be efficiently utilised for succinic acid production.     

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.     

Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of α-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High α-amylase yield of 15,833 U g⁻¹ dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32°C for 54 h whereas α-amylase production in SSF reached its maxima (12,899 U g⁻¹ dry solid ) at 30°C after 66 h of incubation. With the supplementation of 1% NaNO₃, the maximum activity obtained was 19,665 U g⁻¹ dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g⁻¹ dry solid in presence of 0.1% Triton X-100 (20% higher than the control).     

Numerical simulation and PEPT measurements of a 3D conical helical-blade mixer: a high potential solids mixer for solid-state fermentation

Helical-blade solids mixers have a large potential as bioreactors for solid-state fermentation (SSF). Fundamental knowledge of the flow and mixing behavior is required for robust operation of these mixers. In this study predictions of a discrete particle model were compared to experiments with colored wheat grain particles and positron emission particle tracking (PEPT) measurements. In the discrete particle model individual movements of particles were calculated from interaction forces. It was concluded that the predicted overall flow behavior matched well with the PEPT measurements. Differences between the model predictions and the experiments with wheat grains were found to be due to the assumption that substrate particles were spherical, which was in the model. Model simulations and experiments with spherical green peas confirmed this. The mixing in the helical-blade mixer could be attributed to (1) the transport of particles up and down in the interior of the mixer, and (2) dispersion or micro-mixing of particles in the top region of the mixer. It appeared that the mixing rate scaled linearly with the rotation rate of the blade, although the average particle velocity did not scale proportionally. It may be that the flow behavior changes as a function of the rotation rate (e.g., changing thickness of the top region); further study is required to confirm this. To increase the mixing performance of the mixer, a larger blade or a change in the shape of the mixer (larger top surface/volume ratio) is recommended.     

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.     

Effect of hydrolytic and oxidative enzymes produced by solid-state fermentation on greige linen fabric

Solid state fermentation (SSF) was applied for production of fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens using cotton seed-coat fragment waste as a carbon source and enzyme inducer. Lignin-holocellulose matrix of cotton seed coat fragment proved to be effective in inducing production of ligninolytic, cellulolytic and xylanolytic enzymes in solid-state fermentation. The effect of the enzymes produced by SSF on greige linen fabric is discussed and evaluated. In the first experiment the hydrolytic and accompanying oxidative enzymes in the buffer extract of the whole SSF cultures were used for fabric treatment. In the second trial, the enzymes produced in situ (whole SSF material—mixture of fungal biomass, residual substrate and enzymes) were used for the treatment. Weight loss, reducing sugar liberation and removal of colouring materials were measured. The results showed that at equal enzyme charges the intact SSF materials were more efficient than the enzyme extracts. Of the six strains evaluated, Ph. chrysosporium VKM F-1767 was the most effective in removing colouring matters from greige linen fabric.     

Tannase production by Aspergillus aculeatus DBF9 through solid-state fermentation

Tannase an industrially important enzyme was produced by Aspergillus aculeatus DBF9 through a solid-state fermentation (SSF). The organism produced good amount of enzyme and gallic acid in wheat bran among the solid substrate used in SSF. Maximum enzyme and gallic acid production occurred in 5% tannic acid after 72 h. Eighty percent initial substrate moisture and 30 degrees C temperature was found suitable for tannase production.     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

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 the Virtual Instrument (VI) concept, the performance of the 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. The text was submitted by the authors in English.