Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.     

Influence of some environmental factors on Rhizopus nigricans submerged growth in the form of pellets

Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH below 4 and above 7, the presence of Ca²⁺ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was mainly influenced by the inoculum size and the presence of baffles and Ca²⁺ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation, resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of pellet size and pellet quality on pig performance

An experiment involving 1360 growing finishing pigs was undertaken to examine the effect of pellet size and pellet quality, as measured by the Holmen pellet durability test, on pig performance. A barley and soya bean meal diet was used and pellets were of two sizes, 5 and 10 mm diameter. Pellet quality was varied to give two types of pellet by steam conditioning and screening procedure during the pelleting process. The mean difference obtained in pellet durability was 11%.

Pig performance between 30 and 80 kg liveweight was not affected by either pellet size or pellet quality. There were small non-significant trends in favour of both the smaller pellets and the lower quality pellets. These trends, of the order of 1% or less, followed the same pattern as the dry matter content of the diets. The smaller diameter pellets were dried more efficiently in the cooling process and the low durability pellets had less steam added during the manufacturing process, which was reflected in the dry matter content of the finished diets.     

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.     

A mathematical model for the growth of mycelial pellet populations

In liquid culture, filamentous organisms often grow in the form of pellets. Growth result in an increase in radius, whereas shear forces result in release of hyphal fragments which act as centers for further pellet growth and development. A previously published model for pellet growth of filamentous microorganisms has been examined and is found to be unstable for certain parameter values. This instability has been identified as being due to inaccuracies in estimating the numbers of fragments which seed the pellet population. A revised model has been formulated, based on similar premises, but adopting a finite element approach. This considers the population of pellets to be distributed in a range of size classes. Growth results in movement to classes of increasing pellet size, while fragments enter the smallest size class, from which they grow to form further pellets. The revised model is stable and predicts changes in the distribution of pellet sizes within a population growing in liquid batch culture. It considers pellet growth and death, with fragmentation providing new centers of growth within the pellet population, and predicts the effects of shear forces on pellet growth and size distribution. Predictions of pellet size distributions are tested using previously published data on the growth of fungal pellets and further predictions are generated which are suitable for experimental testing using cultures of filamentous fungi or actinomycetes. (c) 1995 John Wiley & Sons, Inc.     

Enhanced enzyme production with the pelleted form of <Emphasis Type="Italic">D. squalens</Emphasis> in laboratory bioreactors using added natural lignin inducer

White-rot fungi are extensively used in various submerged biotechnology processes to produce ligninolytic enzymes. Transfer of the process from the laboratory to the industrial level requires optimization of the cultivation conditions on the laboratory scale. An interesting area of optimization is pellet growth since this morphological form solves problems such as the decreased oxygen concentration, limited heat, and nutrient transport, which usually occur in dispersed mycelium cultures. Many submerged fermentations with basidiomycetes in pellet form were done with Phanerochaete, Trametes, and Bjerkandera species, among others. In our study, another promising basidiomycete, D. squalens, was used for ligninolytic enzyme production. With the addition of wood particles (sawdust) as a natural inducer and optimization of mixing and aeration conditions in laboratory stirred tank (STR) and bubble column (BCR) reactors on pellet growth and morphology, the secretion of laccase and the manganese-dependent peroxidase into the medium was substantially enhanced. The maximum mean pellet radius was achieved after 10 days in the BCR (5.1 mm) where pellets were fluffy and 5 days in the STR (3.5 mm) where they were round and smooth. The maximum Lac activity (1,882 U l⁻¹) was obtained after 12 days in the STR, while maximum MnP activity (449.8 U l⁻¹) occurred after 18 days in the BCR. The pellet size and morphology depended on the agitation and aeration conditions and consequently influenced a particular enzyme synthesis. The enzyme activities were high and comparable with the activities found for other investigations in reactors with basidiomycetes in the form of pellets.     

Production of the ligninolytic enzymes by immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in an air atmosphere

Summary The production of the ligninolytic enzymes by Phanerochaete chrysosporium immobilized on polyurethane foam cubes in air was investigated by adopting different sizes and amounts of the carriers, different medium C/N ratios and different glucose-feeding strategies. No lignin peroxidase (LiP) activity was observed under nitrogen limitation (C/N ratio, expressed as glucose/NH₄⁺, 56/2.2 mM) with two sizes and three amounts of the carriers, while comparable levels of manganese peroxidase (MnP) activities were detected only in non-immersed cultures with two sizes of the carriers. A non-immersed state also stimulated LiP formation under carbon limitation (C/N ratio 28/44 mM). High peak activities of LiP, 197 and 164 U/l, were obtained in non-immersed cultures under carbon limitation at the C/N ratios of 28/44 and 56/44 mM, respectively, the occurrence of the activities coinciding with the complete consumption of glucose. A very low level of MnP was measured at the C/N ratio of 28/44 mM compared with the similar activities at 56/2.2 and 56/44 mM. An addition of 2 g glucose/l after its complete depletion improved both the production of LiP and MnP markedly in non-immersed culture at the initial C/N ratio of 28/44 mM, whereas a replenishment of 5 g/l, still enhancing the formation of MnP, inhibited the production of LiP first before the later reactivation. It is suggested that non-immersed liquid culture under carbon limitation reinforced by a suitable glucose feeding strategy is one potential way to realize high production of the ligninolytic enzymes by P. chrysosporium in air.     

Perfusion cultures and modelling of oxygen uptake with three-dimensional chondrocyte pellets

Chondrocyte pellets were cultivated in a perfused flow chamber and supplied with medium by a constant flow rate from a conditioning vessel. In this conditioning vessel the medium was aerated and used medium was exchanged semi-continuously. The higher amount of DNA and glycosaminoglycane (GAG) in these pellets compared to control cultures under stationary conditions showed a positive effect of the reactor system, compared to standard culture conditions. A diffusion reaction model was applied to calculate the oxygen uptake of the cell pellet and to describe the oxygen profile within the pellet. The model included diffusion within the cell pellet and oxygen uptake of the cells. Calculated data were compared to experimental data obtained by tissue engineered chondrocyte cell pellets. Model calculations agreed rather well with experimental data.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

A Feasibility Study on Pellet Coating Using a High-Speed Quasi-continuous Coater

Pellet coating is traditionally carried out using the Wurster coater. This study investigated the feasibility of pellet coating in a newly developed coater built with a unique airflow system, the Supercell™ coater (GEA Pharma Systems, UK). A full factorial design study was carried out to evaluate the influences of the spray rate of the coating dispersion, batch size of the pellet load, pellet size fraction and plenum pressure of the fluidizing air on the color coating of pellets in the Supercell™ coater. Results showed that pellets could be successfully coated using the Supercell™ coater. Higher plenum pressures and lower spray rates were found to minimize pellet agglomeration during coating. Although coating efficiencies were comparable amongst the different pellet size fractions, larger batch sizes of pellets were coated with higher efficiencies. Process optimization was carried out for each pellet size fraction and a large batch size (120 g) in combination with a high plenum pressure (1,500 mm WC) were deemed optimal. Optimal spray rates differed according to pellet size fraction and a lower spray rate was required for smaller pellets. Pellet flow patterns observed during coating were dependent on the pressure drop across the fluidized load. A ‘swirling’ pellet flow pattern was generally observed at coating conditions which led to optimal outcomes.KEY WORDS: fluid bed, fluidization, pellet coating, pellet flow patterns, pressure drop, process optimization, Supercell™ coater     

Total ammonia nitrogen leaching from feed pellets used in salmon aquaculture

The total amount of ammonia nitrogen (TAN) leaching from Atlantic salmon ( Salmo salar L.) feed pellets was determined under controlled laboratory conditions. The leaching was successfully explained by means of a first order kinetic equation:     , where a was the maximum TAN leached and k was the velocity of the process. Then t _a was defined as the time at which a was reached, which was obtained from the equation prediction. The interaction between pellet size and immersion length significantly influenced TAN leaching: the smaller the pellet and the longer the immersion times, the higher the leaching ( a ). However, pellet size did not influence k or t _a , although k tended to be higher and t _a lower for larger pellets. TAN leaching from feed pellets depends on the protein content and contact surface. As the surface/volume ratio (S/V) increases with pellet size while the protein content decreases, it was hypothesized that S/V influences k whereas the protein content has a greater effect on a .     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Influence of operational variables on properties of piroxicam pellets prepared by extrusion-spheronization: A technical note

Summary and Conclusion  The processing conditions has a pronounced effect on the pellet properties. Drying conditions influenced the mean size and the drug release of the pellets. Because of the shrinking of the pellets upon drying at higher temperatures, the pellets also showed increased densities. Freeze drying almost prevented shrinking and thus led to the highest drug release. With an increase in the temperature of drying, the drug release rate decreased. Both spheronization time and spheronization speed affected the shapes of pellets, and the changes in shapes then affected the pellet flow properties. Within the studied range, the circularity of the pellets was affected more by the spheronization time than by the spheronization speed. Drying conditions influenced pellet friability, which decreased with an increase in drying temperature, indicating the formation of more dense structures at higher temperatures. The same result was obtained with spheronization time. With an increase in spheronization time, the friability decreased, because of the formation of more compact masses at higher spheronization time. Mean size was not affected by spheronization time or spheronization speed. Published: March 9, 2007     

Agitation effects on submerged growth and product formation of Aspergillus niger

Product formation of mycelial organisms, like Aspergillus niger, is intimately connected with their morphology. Pellet morphology is often requested for product formation. Therefore, it is important to reveal the influence of the hydrodynamic conditions on the morphological development. In the present study, pellet morphology and glucoamylase formation were studied under different agitation intensities of A. niger AB1.13. For pellet formation inside the bioreactor, without the use of precultures, it is necessary to work at low energy dissipation rates. Biomass growth and glucoamylase activity were correlated with energy dissipation. Furthermore, product yield was analysed in dependence of pellet size and concentration. The present work shows that simple equations based on Monod-kinetics can describe growth and product formation, in general, also in mycelian organisms. All measured morphological data, like pellet concentration, as well as glucoamylase formation, strongly depend on the hydrodynamic conditions.     

Ibuprofen-Loaded Calcium Stearate Pellets: Drying-Induced Variations in Dosage Form Properties

Pellets intended for oral dosing are frequently produced via extrusion/spheronization followed by drying. Typically, the last active process step, i.e., drying, is assumed to have little effect on the final dosage form properties (e.g., dissolution characteristics). Thus, there exist only a few studies of this subject. In the present study, calcium stearate/ibuprofen pellets were used as model system to investigate the impact of the drying conditions. Lipophilic calcium stearate matrix pellets containing 20% ibuprofen were prepared via wet extrusion/spheronization. Subsequently, desiccation, fluid-bed drying, and lyophilization were applied for granulation liquid removal. The impact of these drying techniques on the final pellet properties was evaluated. The in vitro dissolution behavior was dramatically altered by the drying techniques that were considered. The investigated pellets showed drug release rates that varied as much as 100%. As no polymorphic transitions occurred during drying, we focused on two possible explanations: (a) a change in the drug distribution within the pellets and (b) a change in pellet micro-structure (porosity, pore size). The ibuprofen distribution proved to be homogeneous regardless of the drying conditions. Pellet porosity and pore sizes, however, were modified by the drying process. Our results clearly demonstrate that a single process step, such as drying, can play a crucial role in achieving desired pellet properties and release profiles.     

Seasonal changes and altitudinal variation in deer fecal pellet decay

Surveying dung pellet groups to estimate population size requires knowledge of the dung decay rates in different environmental conditions. We evaluated seasonal and elevational differences in the decay rates of fresh deer pellets in the Ashio–Nikko Mountains, Japan. At each of four sites (1,500, 1,200, 900, and 600 m above sea level), we set out four replicates of 50 fresh deer pellets each month from September 2008 to August 2009. Dung pellet groups were evaluated after 24 h and again monthly. We also monitored the dung beetle fauna monthly using pitfall traps. Dung beetle activity was positively correlated with temperature, and the rate of dung decay was strongly correlated with dung beetle dry weight. Decay was most rapid during summer and at lower altitudes; pellets set out during winter did not decay until the following spring. Resource managers who use dung surveys to estimate population sizes in mountainous regions should consider the decay rates at different altitudes.     

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.     

Survival and growth in groups of a subsocial spider (Stegodyphus lineatus)

Summary In spiders, known as potentially cannibalistic, mutual tolerance is one important requirement for group life. Using the subsocial spiderStegodyphus lineatus which possibly resembles the ancestors of the social species, the effects of competition were investigated in the laboratory. When dispersal was prevented, spiderlings were capable of living in groups. The intensity of competition for food among spiders in groups was varied experimentally by varying group size or the relative size differences of individuals. Body mass and mortality were compared in the different experiments. Prey availability, the size of the spiders and initial body size differences among group members all influenced the survival probability and growth of the spiders. Spiders of equal size tolerated each other with a higher probability than spiders of different sizes. Feeding in groups was always disadvantagous even for the largest spiders.     

Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes

The morphological type of a microorganism generally influences its metabolite production. In the present study, we investigated the effects of the mycelial morphology of shiitake (Lentinula edodes) on the production of 2-mercaptohistidine trimethylbetaine (ergothioneine, ESH) during liquid fermentation. Analyses of the distribution of ESH in mycelial cells of different morphological types revealed that the ESH content of pellets obtained from the liquid fermentation media was much greater than the content in the free mycelia and clumps. The concentration of ESH in pellets on day 15 of liquid fermentation reached 0.79 mg/g dry weight (DW), which is approximately three times the concentration found in mycelia clumps (0.28 mg/g DW) and free mycelia (0.31 mg/g DW). Macroscopic image analysis of the development and morphological changes of the pellets during a liquid fermentation period of up to 25 days indicated that pellet growth showed a highly positive correlation with the increase in ESH concentration (r ² = 0.9851). A reduced agitation rate of 50 rpm for the culture medium was suitable for pellet formation and size enlargement. The results obtained in this work would be helpful in guiding the intentional manipulation of the distribution and enrichment of ESH in L. edodes through changes in liquid fermentation conditions.     

Modeling and measurements of fungal growth and morphology in submerged fermentations

Generalizing results from fungal fermentations is difficult due to their high sensitivity toward slight variation in starting conditions, poor reproducibility, and difference in strains. In this study a mathematical model is presented in which oxygen transfer, agitation intensity, dissolved oxygen tension, pellet size, formation of mycelia, the fraction of mycelia in the total biomass, carbohydrate source consumption, and biomass growth are taken into account. Two parameters were estimated from simulation, whereas all others are based on measurements or were taken from literature. Experimental data are obtained from the fermentations in both 2 L and 100 L fermentors at various conditions. Comparison of the simulation with experiments shows that the model can fairly well describe the time course of fungal growth (such as biomass and carbohydrate source concentrations) and fungal morphology (such as pellet size and the fraction of pellets in the total biomass). The model predicts that a stronger agitation intensity leads to a smaller pellet size and a lower fraction of pellets in the total biomass. At the same agitation intensity, pellet size is hardly affected by the dissolved oxygen tension, whereas the fraction of mycelia decreases slightly with an increase of the dissolved oxygen tension in the bulk. All of these are in line with observations at the corresponding conditions.