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     

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.     

A computer model for the growth and differentiation of a fungal colony on solid substrate

Mold growth and differentiation are closely related to the formation of secondary products. In solid-substrate fermentations this interrelationship is often more completely realized than in submerged cultures. Solid substrate reactions are used commercially in a limited manner in the western world, but are relatively common in Asia. Basic studies in solid-substrate fermentation should yield results applicable to all types of commercial mold fermentations for the production of a secondary product. This paper presents a relatively simple model for the growth of a mold colony on a solid surface with a defined medium utilizing glucose. Unlike submerged cultures the model must account for both cellular differentiation and the spatial heterogeneity in the system. Model parameters were estimated independently using literature values. The results of the simulation studies suggest that mass transfer limitations are at least partially responsible for the proliferation of differentiated structures on solid substrates as compared to liquid cultures. Since the concentration profile depends on the depth of the substratum, conditions that enhance conidia production can be achieved by controlling the depth of the solid medium.     

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.     

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.     

Two-phase model of the kinetics of growth of Rhizopus oligosporus in membrane culture

An empirical model was developed to describe a growth profile occurring in solid-state fermentation (SSF), namely that consisting of an initial period of rapid acceleration followed by an extended period of deceleration. This kinetic profile is not adequately described by the logistic model. The empirical model is based on the concept of active and nonactive hyphal segments. Exponential and deceleration growth phases are modeled. The model parameters can be determined directly from the dry-weight profile and they depend on the growth medium present. The model suggests that, at the instant the culture enters the deceleration phase, there is a 71% to 86% decrease in the number of actively extending hyphal tips and that, during the deceleration phase, there is an exponential decay in the number of active hyphal segments, with a first-order decay constant of 0.042 to 0.072 h(-1).     

Macroscopic growth of filamentous fungi on solid substrate explained by a microscopic approach.

A quantitative model predicting biomass growth on solid media has been developed. The model takes into account steric interactions between hyphae and tips at the microscopic level (competition for substrate and tip-hypha collisions). These interactions effect a slowing down of the hyphal, population-averaged extension rate and are responsible, at the microscopic level, for the distribution of tip orientations observed at the colony border. At the macroscopic level, a limiting value of the colony radial extension rate is attained. A mathematical model that combines hyphal branching, tip diffusion, and biomass growth was proposed to explain such behavior. Experiments using Gibberella fujikuroi were performed to validate the model; good agreement between experiments and simulations was achieved. Most parameters can be measured by simple image analysis on the peripheral growth zone, and they have clear physical meaning; that is, they correspond to properties of single, leading hyphae. The model can be used to describe two-dimensional (2D) solid media fermentation experiments under varying culture conditions; the model can also be extended to consider growth in three-dimensional (3D), complex geometry substrates.     

Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt (-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates.     

Simple dissolution-reaction model for enzymatic conversion of suspension of solid substrate

Although reactions in substrate suspension are employed in industry for several bioconversion processes, there appears to be no quantitative model available in the literature to rationalize the optimization of these processes. We present a simple model that incorporates the kinetics of substrate dissolution and a simultaneous enzymatic reaction. The model was tested in the alpha-chymotrypsin-catalyzed hydrolysis of an aqueous suspension of dimethyl benzylmethylmalonate to a homogeneous solution of enantiomerically pure monoester. This reaction occurs in the bulk phase, so catalysis by enzyme absorbed at the solid-liquid interface plays no role. The value of the parameters in the model (i.e., the mass transfer coefficient of substrate dissolution (k(L)), the substrate solubility, and the rate constant for the enzymatic reaction) were determined in separate experiments. Using these parameter values, the model gave a good quantitative prediction of the rate of the overall dissolution-reaction process. When the particle size distribution is known, k(L) may also be calculated instead. The model seems to be applicable also for other poorly soluble substrates, other enzymes, and other solvents. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 433-440, 1997.     

A kinetic model for microbial growth on solid hydrocarbons

A kinetic model has been presented to explain the growth of microorganism on solid hydrocarbons. The model is based on the assumption that metabolite produced by the growing cells helps the dissolution of the solid substrate in the aqueous medium. The linear behavior of the growth curve predicted by the model is verified experimentally.     

Influence of the water activity of a solid substrate on the growth rate and sporogenesis of filamentous fungi

The water activity (a(w)) of substrates has been related to the mycelial growth and the sporogenesis of two molds. In the absence of other limiting factors, optimal a(w) values were determined for growth and sporogenesis as 0.99 and 0.98, respectively, for Trichoderma viride TS and 0.97 and 0.96 for Penicillium roqueforti. In all cases, the accuracy of the optimal value would justify the regulation of this parameter. A model was proposed which establishes a relationship between the mycelial growth and the water activity value of the substrate.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.     

A computational approach to model gliding motion of an organism on a sticky slime layer over a solid substrate

Biomechanics and Modeling in Mechanobiology - Bacteria are microscopic single-celled microbes that can only be spotted via a microscope. They occur in a variety of shapes and sizes, and their...     

Automation of a solid substrate cultivation pilot reactor

An automation system for a solid substrate pilot bioreactor is described. The performance of the system in real time experiments is discussed and future improvements are proposed. Good control of temperature and water content of the solid bed was achieved, although the system is not fully automatic and needs human supervision.     

Use of sugarcane bagasse pith as solid substrate forP. chrysosporium growth

Phanerochœte chrysosporium strain H-298 grown on sugarcane bagasse pith, a lignocellulosic residue, is proposed as a bioremediation agent for aromatic contaminated soils. To investigate the use of pith for the development of a fungal inoculum, the effect of culture conditions on fungus survival and microbial respiration under solid fermentation were studied. Microbial respiration, estimated from the CO₂ evolution rates, was maintained relatively high at low aeration conditions. High respiration occurred in cultures with 2,2-dimethylsuccinate added and without buffers, but not in those with acetate, succinate and phosphate buffers. It was observed that the culture was autobuffered at pH 4.5, due to acetic acid release, and that moisture content increased from 60 to 70%; these conditions were appropriate for fungal cultivation. CO₂ evolution rates and fluorescence analysis showed that fungal survival was maintained through 18 d.     

Ergosterol content of Rhizopus oligosporus NRRL 5905 grown in liquid and solid substrates

Summary The ergosterol content of Rhizopus oligosporus NRRL 5905 varied between 2–24 g/ mg biomass dry matter when grown in laboratory media and was found to be influenced by the substrate composition.When grown on a natural substrate (soya beans) the ergosterol content was considerably higher (estimated at approx. 60–90 g/mg biomass dry matter).In laboratory media, the ergosterol content was also influenced by the extent of aeration and the growth phase of the mycelium; within the range of 25° C–35° C, the incubation temperature did not influence the ergosterol content significantly.In view of these variations, ergosterol should not be used as a chemical index for the quantification of biomass grown in static solid-substrate fermentations with limited mass transfer, e.g. tempe or oncom.