The morphology of filamentous fungi.

The morphology of fungi has received attention from both pure and applied scientists. The subject is complicated, because many genes and physiological mechanisms are involved in the development of a particular morphological type: its morphogenesis. The contribution from pure physiologists is growing steadily as more and more details of the transport processes and the kinetics involved in the morphogenesis become known. A short survey of these results is presented. Various mathematical models have been developed for the morphogenesis as such, but also for the direct relation between morphology and productivity--as production takes place only in a specific morphological type. The physiological basis for a number of these models varies from thorough to rather questionable. In some models, assumptions have been made that are in conflict with existing physiological know-how. Whether or not this is a problem depends on the purpose of the model and on its use for extrapolation. Parameter evaluation is another aspect that comes into play here. The genetics behind morphogenesis is not yet very well developed, but needs to be given full attention because present models and practices are based almost entirely on the influence of environmental factors on morphology. This makes morphogenesis rather difficult to control, because environmental factors vary considerably during production as well as on scale. Genetically controlled morphogenesis might solve this problem. Apart from a direct relation between morphology and productivity, there is an indirect relation between them, via the influence of morphology on transport phenomena in the bioreactor. The best way to study this relation is with viscosity as a separate contributing factor.

A particulate pulse-release system and mathematical description with the Maxwell-Stefan theory.

In this contribution both the development of a multi-particulate delayed release system with release properties dependent on the swelling of an UV crosslinked coating and a mathematical model to describe its release properties are presented. The formulation consists of a water-soluble core coated with a copolymer of methacrylic acid and ethyl acrylate. Incorporating a network of crosslinked pentaerythritol triacrylate decreases the water-solubility of the coating. After immersing the formulation in water the coating will take up water and subsequently swell in such a degree that the diffusion coefficient of water in the coating will increase. This makes the coating permeable to the dissolved components present in the core. The swelling kinetics of the coating are such that the formulation has a pulse-release profile, i.e. a fast release of the contents is obtained after a pre-determined lag-time. Both the coating thickness and the duration of the UV crosslinking time can be used to adjust the lag-time. The experimental results are used to estimate the Maxwell-Stefan diffusion coefficients of water in the coating. The relation between the Maxwell-Stefan diffusion coefficient and the mole fraction of water in the coating differs from results found in the literature. However, the prediction of the release time based on the presented model is in good agreement with the experimental findings.

Fluid bed agglomeration with a narrow droplet size distribution.

In the fluid bed agglomeration processes liquid distribution influences the agglomerate growth. We developed a new nozzle that produces uniform droplets, which allows droplets to be easily controlled in size independently of liquid- and airflow of the nozzle. It was found that the spray rate and the mixing in the spray zone determine the average granule size and that there is linear relation between the number of droplets of which a granule consists and its volume, at the early stage of the process. The nucleation ratio factor introduced in this paper depends on the material properties of binder liquid and powder particles and is a useful parameter to describe the binder liquid efficiency. The decline of the growth rate of granules during the agglomeration process was due to the less sufficient rewetting of granules resulting in less growth. A linear relation was found between tracer mass added to the binder liquid and the granule mass in an early stage of the process. Solubility of the tracer was found not to influence its distribution. The new nozzle proves to be a good tool to study the effect of wetting and growth of granules.

Microdialysis of glucose in subcutaneous adipose tissue up to 3 weeks in healthy volunteers.

OBJECTIVE: To measure possible changes in dialysate glucose concentrations over time, to validate the diffusional model for glucose transport from tissue to the probe, and to evaluate the actual glucose concentration in adipose tissue. RESEARCH DESIGN AND METHODS: Glucose concentrations in the subcutaneous adipose tissue of five healthy subjects (age 25 +/- 2.7 years, BMI 23.2 +/- 2.3 kg/m2 [mean +/- SD]) were measured by the microdialysis technique and compared with blood glucose. We applied microdialysis probes with hollow fibers of various membrane length (10-35 mm), used eight perfusion flow rates (0.5-20 microl/min), and perfused four glucose solutions (0.0, 2.8, 8.3, 11.1 mmol/l). RESULTS: After implantation, a substantial decrease in glucose recovery to the lowest value of 26 +/- 10% of the final plateau value was noted during the first few hours (n = 4). Recovery increased and stabilized after 5-9 days at 84.0 +/- 7.4% of capillary blood glucose when a flow rate of 0.5 microl/min was applied. According to the zero net-flux method, the glucose concentration in equilibrium, Cequi, with the surrounding tissue can be obtained. This concentration also decreases; however, 1 h after recovery, Cequi increases again over 1 or 2 days to a stable value that is not significantly different from the measured capillary blood glucose (P < 0.05). Using various perfusion flow rates and probes (membrane length 10-35 mm), it is shown that diffusion is the rate-limiting process for glucose transport through tissue. CONCLUSIONS: Insertion of the microdialysis probes causes damage to the adipose cells and the vascular bed around the probe. Glucose recovery decreases because of a lower blood supply. In 5-9 days, glucose recovery increases; apparently, this time is needed to repair the microstructure of tissue around the probe. After stabilization of the recovery, no loss of probe permeability, which is due to biocompatibility problems, was seen. The change during the 2 days in equilibrium concentration is probably caused by an inflammation reaction that consumes glucose around the probe. The individual increase in recovery during the 1st days after probe insertion until a stable plateau value is reached (flow rate >0 microl/min) is complicated for short-term clinical glucose measurements in adipose tissue. After stabilization, the mean equilibrium concentration of all subjects was equal to the mean capillary blood glucose concentration. Therefore, we conclude that capillary blood glucose concentration probably is the driving force for diffusion through the capillary wall into the probe and is not some interstitial concentration.

Experimental simulation of oxygen profiles and their influence on baker's yeast production: I. One-fermentor system.

In production-scale bioreactors microorganisms are exposed to a continually changing environment. This may cause loss of viability, reduction of the yield of biomass or desired metabolites, and an increase in the formation of by-products. In fed-batch production of baker's yeast, profiles may occur in substrate and oxygen concentrations and in pH. This article deals with the influence of a periodically changing oxygen concentration on the growth of baker's yeast in a continuous culture. Also, influences on the production of ethanol, glycerol, acetic acid, and on the composition of the cells were investigated. It was found that relatively fast fluctuations between oxygen-unlimited and oxygen-limited conditions with a frequency of 1 or 2 min had a distinct influence on the biomass and metabolite production. However, RNA, protein, and carbohydrate contents measured in cells exposed to fluctuations differed little from those in cells from an oxygen-unlimited or an oxygen-limited culture. The respiration and fermentation capacities of cells exposed to fluctuations can be larger than the capacities of cells grown under oxygen-unlimited conditions.

Experimental simulation of oxygen profiles and their influence on baker's yeast production: II. Two-fermentor system.

A reactor configuration consisting of two reactors with an exchange flow was used for the experimental simulation of large-scale conditions. The influence of fluctuations in oxygen concentration on the growth and metabolite production of baker's yeast was investigated by sparging one fermentor with air and one with nitrogen gas. It was found that the biomass yield decreased and the metabolite formation increased with rising circulation time (longer oxygen-unlimited and oxygen-limited periods). Not only was the performance of the oxygen-limited fermentor characterised by (partly) reductive metabolism, but that of the oxygen-unlimited fermentor as well. The results of the experiments in this reactor system were compared with those from the experiments carried out in a one-fermentor system with periodically changing oxygen concentrations. The formation of acetic acid, which is characteristic for transient states, showed a distinct difference between the two reactor systems.

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.

An integral dynamic model for the UASB reactor.

In this article a dynamic model of a continuous working UASB reactor is described. It results from the integration of the fluid flow pattern in the reactor, the kinetic behavior of the bacteria (where inhibition and limitation were taken into account), and the mass transport phenomena between different compartments and different phases. The mathematical equations underlying the model and describing the important mechanisms were programmed and prepared for computations and simulations by computer. The settler efficiency has to be over 99% to prevent the reactor from wash-out. When the settler efficiency is over 99%, the total sludge content of the reactor increases steadily, so the reactor is hardly ever in a steady state. This implies dynamic modeling. The model is able to predict the various observable and nonobservable or difficult to observe state variables, e.g., the sludge bed height, the sludge blanket concentration, the short-circuiting flows over bed and blanket, and the effluent COD concentration as a function of the hydrodynamic load, COD load, pH, and settler efficiency. The optimal pH value is between 6.0 and 8.0; fatty acid shock loadings are difficult to handle outside this optimal pH range.

Kinetics of anaerobic purification of industrial wastewater.

As a part of the development of an integral mathematical model describing the up-flow anaerobic sludge blanket (UASB) reactor, the kinetics of the conversion of organic wastes has to be known. We compared the Monod model with the model proposed by Andrews et al. Together with the assumption that the substrate for the anaerobic bacteria is formed by nonionized, volatile fatty acids, the Andrews model is able to describe substrate inhibition and reactor failure due to pH changes.From four batch experiments, with different concentrations of microorganisms, it could be concluded with a reliability of over 95% that the monod model was inadequate and Andrews' model was adequate to describe the measurements. Standard statistical techniques like the X2- and the F-test were used for this purpose. From a parameter sensitivity analysis for the Andrews model it followed that the maximum specific growth rate microAmax of the bacteria and the inhibition constant K1 are the parameters which influence the system most. Thus, these parameter were determined experimentally and most accurately. The results are: microAmax =16*10-4h-1+/-2% and K1 = 0.0158 g HAc/L+/-2.5%The other parameters were taken from literature. From calculation of the Thiele modulus for the particles it follows that transport limitation of the substrate in the flocs is not significant. The efficiency is 0.85 in the worst case..

Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans.

To simulate production-scale conditions of gluconic acid fermentation by Gluconobacter oxydans, different experimental setups are presented in this study. From the determination of the time constants of a production-scale reactor, it can be concluded that mixing and oxygen transfer are the rate-limiting mechanisms. This results in oxygen concentration gradients which were simulated in a one-compartment reactor in which the oxygen concentration was fluctuated by a fluctuated gassing with air and nitrogen. It could be concluded that only very long periods of absence of oxygen (ca. 180 s) results in lower specific oxygen uptake rates by Gluconobacter oxydans. From scale-down studies carried out in a two-compartment system to simulate a production-scale reactor more accurately, it could be concluded that not only the residence time in the aerated part of the system is important, but the liquid flow in between the different parts of the reactor is also an essential parameter. It could also be concluded that the microorganisms are not influenced negatively by the fluctuated oxygen concentrations with respect to their maximal oxidation capacity. The two-compartment system can also be used for optimization experiments in which the "aerated" compartment was gassed with pure oxygen. From these experiments it was concluded that also a short residence of the cells at high oxygen concentrations diminished the growth and product formation rates. These experiments show the necessity of the scale-down experiments if optimization is carried out. The two-compartment system presented in this study is a very attractive tool for reliable scale-down experiments.

Theory and applications of unstructured growth models: Kinetic and energetic aspects.

Microbial kinetics and energetics are discussed in connection with the formulation of unstructured growth models. The development of microbial energetics and the use of macroscopic methods in the study of microbial growth are briefly evaluated. The general approach to the modelling of microbial growth has been critically discussed and a strategy for the formulation of unstructured models is presented. A simple unstructured model based on Monod kinetics and the linear relation for substrate consumption is evaluated with reference to extensive experimental and simulation data obtained in batch, fed-batch, and continuous cultivation modes. Choice for a kinetic expression is discussed and has been shown not to be critical in most situations. It is shown that during growth in batch mode, the behavior of the system is rigidly fixed by the kinetic parameter: the maximum specific growth rate. The energetic parameters have minimal influence. In continuous cultivation the behavior is fixed by the energetic parameters: the maximum yield and the coefficient of maintenance. Implications of these observations have also been discussed. The linear relation for substrate consumption is tested with continuous culture data. It is shown that significant deviations at low growth rates cannot be fully accounted by the loss of viability. The situations where unstructured models will be adequate or not for system description, are evaluated and checked experimentally. Influence of an environmental factor, the temperature, on the unstructured model parameters is also quantitatively described. It is concluded that the art of unstructured model building has already reached its maturity and that now much effort should be channelled into the development and verification of structured models.

Continuos IBE fermentation by immobilized growing Clostridium beijerinckii cells in a stirred-tank fermentor.

The potential of continuous isopropanol-butanol-ethanol (IBE) fermentation by Ca-alginate-immobilized Clostridium beijerinckii cells in a continuous stirred-tank reactor is investigated. A mathematical model is presented to describe steady-state reactor performance. It appeared to be possible to use the biocatalyst particles repeatedly for successive fermentations (at least three times for a total duration of two months). Reactor productivity was 6-16 times higher than that of a batch fermentation (free cells), while the solvents yield was also increased. Measurements of substrate, product, and biomass concentrations were only partially in agreement with the model; however, a solid basis for further technological developement of the process has been laid.

Modeling of bacterial growth; formulation and evaluation of a structured model.

Models which consider changes in the composition of biomass in response to environmental changes are called Structured models. They provide a more comprehensive description of microbial behavior than unstructured models. Compared with the unstructured modeling efforts, very little has so far been done on the theory and practice of structured model building. In most of the works reported so far, no experimental data were provided, and hence no means of testing the proposed models were offered. Others only reported macroscopic response data and not the cellular composition. In an attempt to fill some of the gaps in this field, in this work, first the general formal approach to structured modeling is developed in matrix notation. Then, a simple two-compartmental model, i.e., a structured model describing the activity of the biomass with two variables, is described. The cell is divided into two fractions, one of which relates to the RNA fraction. The proposed model was then critically evaluated with experimental data, including the RNA data, obtained from fed-batch and continuous-culture experiments. The importance of using cellular structure data for model verification, i.e., RNA data in this case, is shown. Shortcomings and capabilities of the developed model are discussed.

Unstructured model for growth of mycelial pellets in submerged cultures.

An unstructured model is presented to describe growth of mycelial pellets in submerged cultures. This model integrates growth kinetics at the scale of the hyphae with the physical mechanisms of mass-transfer processes at the scale of the pellets and the fermentor. The main elements of the model are biomass, substrate, and oxygen balances for the liquid phase and the pellets. The possible occurrence of oxygen limitation in the pellets is introduced in analogy with catalyst theories by means of an effectiveness factor. To simulate the growth of pellets the model is transferred into a computer program. The model is tested by means of fermentation experiments in a bubble column. Results of the growth experiments compare favorably with the outcome of computer simulations.