Improvement of growth ofRhizopus oligosporus on a model solid substrate

Summary Metal ions and cassava extracts stimulated growth ofRhizopus oligosporus on a model solid substrate. A large improvement in protein content was obtained by simultaneously increasing the nitrogen content and decreasing the particle size of the substrate. No improvement occurred when these conditions were applied to cassava, however, because of the sticky consistency of the cassava after gelatinization.     

Suppression of penetrative hyphae ofRhizopus oligosporus by membrane filters in a model solid-state fermentation system

Summary Membrane filters overlaid on slabs of a model solid substrate enabled recovery of biomass ofRhizopus oligosporus. Although the presence of the membrane filter affects the growth ofRhizopus oligosporus it provides a useful tool for studying solid-state fermentation.     

Cassava starch fermentation pattern ofRhizopus oligosporus

Summary Rhizopus oligosporus UQM 145F was grown on cassava starch medium without a preliminary treatment of starch. The fermentation pattern ofR. oligosporus is shown to be influenced significantly by the medium composition, pH of the growth culture and the size of the spore inoculum. By varying the carbon, nitrogen and mineral salt concentrations, it was found that either glucose accumulated due to excess hydrolysis of starch or single cell protein production became dominant. In the case of the carbon source, increases in the concentration of starch led to higher biomass values, but lower rates of protein production. These observations led to a new formulated medium, resulting in an 82.9% cassava starch utilization and a true protein yield of 5.32 g/100 g initial substrate. The response ofR. oligosporus to inorganic compounds such as (NH₄)₂SO₄, urea, NaCl and MgSO₄ indicates an interesting fermentation control system.

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Resumen Rhizopus oligosporus UQM 145F se hizo crecer en un medio a base de almidón de casava sin que hubiera pasado por un tratamiento previo con almidón. Se demostró que el patrón de fermentación seguido porRh.oligosporus estaba significativamente influenciado por: la composición del medio, su pH y el tamaño del inóculo. Al variar las concentraciones de carbono, nitrógeno y sales minerales se observó que predominaba la acumulación de glucosa o bien la producción de proteínas unicelulares. Un incremento en la fuente de carbono aumentó la biomasa pero disminuyó el ritmo de producción de proteínas. Estas observaciones permitieron elaborar un nuevo medio de cultivo que resultó en la utilización del 82.9% del almidón de casava con un rendimiento en proteínas de 5.32 g/100 g de sustrato inicial. La respuesta deRh. oligosporus a compuestos inorgánicos como sulfato amónico, urea, cloruro sódico y sulfato magnésico indican la existencia de un sistema de control de la fermentación muy interesante.

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Résumé On a fait croîtreRhizopus oligosporus UQM 145 F sur un milieu à base d'amidon de manioc sans traitement préalable de l'amidon. Le profil de fermentation parR. oligosporus apparaît être influencé de manière significative par la composition du milieu, le pH de la culture en croissance et la taille de l'inoculum de spores. En variant les concentrations en carbone, en azote et en sels minéraux, on remarque la prédominance tantôt de la protéine unicellulaire, tantôt de l'accumulation de glucose. Un accroissement en concentration conduit à des valeurs plus élevées de la biomasse mais en une moindre production de protéines. Ces observations ont abouti à une nouvelle formulation du milieu avec pour résultat une utilisation à 82.9% de l'amidon de manioc et un rendement vrai en protéines de 5.32 g/100 g de substrat initial. La réponse deR. oligosporus aux composés inorganiques comme le sulfate d'ammonium, l'uréi, le chlorure de sodium et le sulfate de magnésium sont indicateurs d'un système très intéressant de contrôle de la fermentation.

     

A semimechanistic mathematical model for growth of Rhizopus oligosporus in a model solid-state fermentation system

A Semimechanistic mathematical model is developed which describes the growth of Rhizopus oligosporus in a model solid-state fermentation system. Equations are presented for the release of glucoamylase, the diffusion of glucoamylase, the hydrolysis of starch, the generation and diffusion of glucose, and the uptake of glucose and conversion into new biomass. Good agreement of the model with the experimental data was obtained only after the glucoamylase diffusivity and the maximum specific glucose uptake rate were altered from their originally determined values. The model recognizes the distributed nature of the solid-state fermentation and therefore is able to predict the concentration profiles of the system components within the substrate. The model provides an insight into the possible rate-limiting steps in solid-state fermentation-the generation of glucose within the substrate and the resulting availability of glucose at the surface.     

A model substrate for solid-state fermentation

Summary A model substrate consisting of cassava starch embedded in kappa-carrageenan was used to mimic the growth ofRhizopus oligosporus on cassava tubers. Growth on the model substrate was similar to that during solid-state fermentation of the actual cassava. However, protein production and starch utilization were slower on the model substrate.     

Protease production by Rhizopus oligosporus in solid-state fermentation

Rice bran was superior to other proteinaceous substrates for protease production by Rhizopus oligosporus ACM 145F in solid-state fermentation. Maximum protease yield was after 72 h. The optimal initial moisture content was 47% (a _w=0.97). Dried, ground and resuspended fermented rice was the most pratical and effective inoculum preparation, although, in the laboratory, spore suspensions prepared directly from agar slants were more convenient. Inoculum density (from 10² to 10⁷ spores/g substrate) and age (3, 5, 7 and 9 days) had little effect on protease yield.The authors are with the Department of Chemical Engineering, The University of Queensland, Brisbane, Queensland, 4072, Australia     

Response of Rhizopus oligosporus to temporal temperature profiles in a model solid-state fermentation system.

Membrane overcultures of Rhizopus oligosporus were shifted from 37 to 50 degrees C for 10 h and then returned to 37 degrees C, mimicking the temporal temperature profiles which typically occur in SSF due to heat transfer limitations. Analysis with a modified two-phase growth model suggests that the temperature upshift causes a 48% decrease in the number of actively extending hyphal tips, and that the first order death rate constant of tips increases from 0. 059 to 0.073 h(-1). The fungus did not immediately recover when the temperature was returned to 37 degrees C. The model assumed that the specific growth rate constant microgram(g) was not affected by the increase of temperature, although contradictory data was obtained from radial growth rate experiments.     

Effect of oxygen and carbon dioxide on germination and growth ofRhizopus oligosporus on model media and soya beans

 The microcolony technique enables the effects of several atmospheric conditions on fungal growth to be studied by measuring the radius of the colony, while excluding effects of those conditions on germination of the sporangiospores. Various concentrations of oxygen and carbon dioxide in the gas environment were found to influence growth of Rhizopus oligosporus on malt extract/soya peptone/agar. The maximum radial growth rate was 1.48 mm/h and the maximum specific growth rate was 0.109 h^-1 at 30 °C. Oxygen became limiting below 1% (v/v), but growth remained possible at levels of 0.001% oxygen. Carbon dioxide stimulated growth at limiting oxygen levels. The specific growth rate increased from 0.043 h^-1 at 0.5% (v/v) oxygen and 0% (v/v) carbon dioxide to 0.096 h^-1 at 0.5% (v/v) oxygen and 5% (v/v) carbon dioxide. A mixture of 0.5% (v/v) oxygen and 35% (v/v) carbon dioxide inhibited growth. Delay of sporangiospore germination due to low (less than 0.001%) amounts of oxygen was not observed with the techniques used. Fungal activity in a rotating drum fermentor was more strongly affected by low levels of oxygen than was biomass formation on model media. High concentrations of carbon dioxide inhibited growth in the rotating drum fermentor at non-limiting levels of oxygen. It is concluded that aeration and heat removal are both essential aspects of optimization of large-scale solid-substrate bioreactors with Rh. oligosporus. Received: 5 August 1994/Received revision: 14 November 1994/Accepted: 5 December 1994     

Agar plate growth studies of Rhizopus oligosporus and Aspergillus oryzae to determine their suitability for solid-state fermentation

Summary Colony radial growth rates of Rhizopus oligosporus and Aspergillus oryzae were compared under various conditions on agar plates containing cassava starch. Both organisms grew well on cassava starch as their sole source of carbon and energy, although growth was stimulated by the addition of yeast extract and peptone. Neither organism utilized ungelatinized starch effectively. The optimum initial pH for R. oligosporus was 7, although good growth was obtained at pH 5 when ammonium sulfate was partially replaced by urea. A. oryzae grew well over a range of initial pH values from 5 to 8. Growth of R. oligosporus was inhibited by NaCl concentrations above 0.5% (w/v) while A. oryzae was unaffected up to 4% NaCl. The best colony radial growth rate obtained for R. oligosporus was 1.01 mm/h, which was far superior to that obtained for A. oryzae (0.29 mm/h). R. oligosporus was chosen as the more suitable organism for future studies of the protein enrichment of cassava by solid-state fermentation.     

Growth ofRhizopus arrhizus in fermentation media

Summary Rhizopus arrhizus biomass attached itself to fermentor walls, baffles and impellers when grown in casein/ glucose media. In shake flasks, dispersed filamentous growth was produced in media containing certain concentrations of glucose and soya flour. Other media tested produced pelleted or clumpy growth. Medium initial pH did not affect morphology type. Dispersed growth could not be obtained by addition of detergents, oils and polymers to a clear glucose/soya peptone medium. Addition of maize solids to this medium resulted in dispersed growth which occurred even in the presence of calcium, which in most media caused pellet formation. Mycelia appeared to bind to the maize particles and use these as growth centres thereby preventing pellet or clump formation. Mycelial pellets appeared to originate either from a single spore or by interaction of branched hyphae from different spores. Medium composition and macro-morphology type correlate with differences in hyphal structures.     

Development of a model solid-state fermentation system

Summary A model solid substrate was developed, consisting of cassava starch and other nutrients embedded in kappa-carrageenan. A suitable fermentation system and analytical techniques were also developed to cope with the constraints imposed by solid-state fermentation.     

Mimicking gas and temperature changes during enzyme production by Rhizopus oligosporus in solid-state fermentation

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 O2 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. © Rapid Science Ltd. 1998     

Use of confocal microscopy to follow the development of penetrative hyphae during growth of Rhizopus oligosporus in an artificial solid-state fermentation system

Two methods were compared for determining the concentration of penetrative biomass during growth of Rhizopus oligosporus on an artificial solid substrate consisting of an inert gel and starch as the sole source of carbon and energy. The first method was based on the use of a hand microtome to make sections of approximately 0.2- to 0.4-mm thickness parallel to the substrate surface and the determination of the glucosamine content in each slice. Use of glucosamine measurements to estimate biomass concentrations was shown to be problematic due to the large variations in glucosamine content with mycelial age. The second method was a novel method based on the use of confocal scanning laser microscopy to estimate the fractional volume occupied by the biomass. Although it is not simple to translate fractional volumes into dry weights of hyphae due to the lack of experimentally determined conversion factors, measurement of the fractional volumes in themselves is useful for characterizing fungal penetration into the substrate. Growth of penetrative biomass in the artificial model substrate showed two forms of growth with an indistinct mass in the region close to the substrate surface and a few hyphae penetrating perpendicularly to the surface in regions further away from the substrate surface. The biomass profiles against depth obtained from the confocal microscopy showed two linear regions on log-linear plots, which are possibly related to different oxygen availability at different depths within the substrate. Confocal microscopy has the potential to be a powerful tool in the investigation of fungal growth mechanisms in solid-state fermentation.     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

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.     

Pineapple waste - a novel substrate for citric acid production by solid-state fermentation

Summary Citric acid production in solid-state fermentation by Aspergillus foetidus ACM3996 was better on pineapple waste than on apple pomace, wheat bran or rice bran. The highest citric acid content achieved on pineapple waste was 16.1 g per 100 g dried pineapple waste, with a moisture content of 70% and in the presence of 3% methanol. This represents a yield of 62.4% based on the sugar consumed.     

Influence of wheat type and pretreatment on fungal growth in solid-state fermentation

The respiration kinetics of Aspergillus oryzae on different varieties of whole wheat kernels were studied. Six wheat varieties were pretreated in two different ways. Five of the six substrates fermented similarly and independently of the pretreatment method. However, pretreatment affected fermentation of one variety of soft wheat (Apollo). T ₂ ¹H-NMR imaging of the water inside the kernels showed a change in water binding inside the kernels when a different pretreatment strategy was used. Differences in free sugar or amino acid content or in kernel stiffness were not significant.     

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.     

Mathematical modeling of Kluyveromyces marxianus growth in solid-state fermentation using a packed-bed bioreactor

This work investigated the growth of Kluyveromyces marxianus NRRL Y-7571 in solid-state fermentation in a medium composed of sugarcane bagasse, molasses, corn steep liquor and soybean meal within a packed-bed bioreactor. Seven experimental runs were carried out to evaluate the effects of flow rate and inlet air temperature on the following microbial rates: cell mass production, total reducing sugar and oxygen consumption, carbon dioxide and ethanol production, metabolic heat and water generation. A mathematical model based on an artificial neural network was developed to predict the above-mentioned microbial rates as a function of the fermentation time, initial total reducing sugar concentration, inlet and outlet air temperatures. The results showed that the microbial rates were temperature dependent for the range 27–50°C. The proposed model efficiently predicted the microbial rates, indicating that the neural network approach could be used to simulate the microbial growth in SSF.     

Realistic model of a solid substrate fermentation packed-bed pilot bioreactor

For any given large-scale solid substrate fermentation (SSF) bioreactor, to assess how well a control system will work in practice requires the most realistic model possible. This model needs to account fully for complicated dynamic reactor behaviour and, in addition, has to include a specific noise model that is capable of reproducing the disturbances observed in SSF bioreactor measurements. In this work, noisy data collected historically from SSF pilot scale fermentations was used to develop such a model. Applying standard signal processing techniques, each measured variable was separated into deterministic and noise signals. Deterministic signals were used to calibrate a previously developed phenomenological model of the bioreactor. Noise signals were used to construct a realistic noise model for each measured variable in turn. Finally, the two models were combined to attain simulations that compared well with real measurements. This integrated model will provide realistic simulations that will prove useful in the design of effective control systems for intermittently mixed SSF bioreactors.