The effect of aeration and agitation on Claviceps purpurea dimorphism and alkaloid synthesis during submerged fermentation

Summary Varying the air flow rate (vvm) in a fermentor under constant drive speed, Claviceps purpurea dimorphism as well as alkaloid biosynthesis were greatly influenced. At a high flow rate (2.5 vvm) sclerotial growth was favoured in seed and in production media, while at a low air flow rate (1.0 vvm) sphacelial growth dominated. When using high flow rates the oxygen uptake rate was small, but at low flow rates it increased markedly. In both cases the alkaloid production was lower than at the intermediate value of 1.5 vvm of air flow rate, which proved to be optimal. This could be explained by the difference in the air/water interface and two-phase oxygen uptake. At a high air/water interface direct oxygen uptake from the gaseous phase prevails, while at a low air/water interface uptake is due to the oxygen liquid-phase only. Thus for optimal fungal development and alkaloid production a compromise between uptake from the liquid and the gaseous phase has to be established by a defined ratio between aeration and agitation.     

A model for oxygen supply in fixed bed reactors with immobilized hybridoma cells

In fixed bed reactors with animal cells immobilized in macroporous carriers sufficient oxygen supply is a critical parameter. For modelling of the oxygen consumption and the oxygen profile in a fixed bed oxygen gradients within the porous carriers and along the length of the fixed bed have to be considered. For the complex geometry of the fixed bed a model structure was assumed, that allows the calculation of the oxygen profile. The model for oxygen supply of the immobilized cells included the transport resistance from the bulk fluid into the carriers and diffusion within the carriers. The model was compared with experimental data obtained with a hybridoma cell line for production of monoclonal antibodies. Model calculations and experimental data agree rather well. The mean volume-specific oxygen uptake rate as an indicator for the cell activity increased with the superficial flow velocity of the bulk liquid flow, and did not depend on the length of the fixed bed in the range tested. This indicates, that the convective transport from the bulk liquid flow between the carriers to the outer surface of the carriers is a dominating transport resistance besides the diffusive oxygen supply within the carriers.     

Model analysis of biological oxygen transfer enhancement in surface-aerated bioreactors

A model was developed to evaluate the effects of cells and surfactants on oxygen transfer in surface-aerated bioreactors. The model assumed the presence of serial layers of adsorbed surfactants and microorganisms directly adjacent to the gas-liquid interface due to their surface activities, followed by a stagnant liquid layer to account for the oxygen transfer resistance in the liquid phase. The interfacial surfactant film, although posing as an additional resistance, was found to have negligible effect on the oxygen transfer rate because of its extremely small thickness as compared to the cell monolayer and the stagnant liquid layer. On the other hand, cells affect oxygen transfer by two mechanisms: the biological enhancement due to the respiration of interfacial cells and the physical blocking resulting from the semipermeable nature of cell bodies. Due to the low specific oxygen uptake rates of the sludges, the two mechanisms were found to be of comparable importance in activated-sludge systems; the oxygen transfer enhancement factor, E, varied from about 0.97 to 1.10 depending on the operating conditions. The biological enhancement effect, however, predominated in fermentations of actively growing bacteria. At relatively low agitation speed (e. g., 300 rpm), the value of E could reach about 3 to 5 in fermentations with high cell concentrations. Effects of other operating variables, such as the agitation intensity, the oxygen content in the mixed liquor, and the bulk cell concentration, on biological oxygen transfer enhancement were also studied. (c) 1992 John Wiley & Sons, Inc.     

Mathematical model for a three-phase fluidized bed biofilm reactor in wastewater treatment

A mathematical model for a three phase fluidized bed bioreactor (TFBBR) was proposed to describe oxygen utilization rate, biomass concentration and the removal efficiency of Chemical Oxygen Demand (COD) in wastewater treatment. The model consisted of the biofilm model to describe the oxygen uptake rate and the hydraulic model to describe flow characteristics to cause the oxygen distribution in the reactor. The biofilm model represented the oxygen uptake rate by individual bioparticle and the hydrodynamics of fluids presented an axial dispersion flow with back mixing in the liquid phase and a plug flow in the gas phase. The difference of settling velocity along the column height due to the distributions of size and number of bioparticle was considered. The proposed model was able to predict the biomass concentration and the dissolved oxygen concentration along the column height. The removal efficiency of COD was calculated based on the oxygen consumption amounts that were obtained from the dissolved oxygen concentration. The predicted oxygen concentration by the proposed model agreed reasonably well with experimental measurement in a TFBBR. The effects of various operating parameters on the oxygen concentration were simulated based on the proposed model. The media size and media density affected the performance of a TFBBR. The dissolved oxygen concentration was significantly affected by the superficial liquid velocity but the removal efficiency of COD was significantly affected by the superficial gas velocity. An erratum to this article can be found online at .     

Modeling of an aerobic biofilm reactor with double-limiting substrate kinetics: bifurcational and dynamical analysis

A mathematical model of an aerobic biofilm reactor is presented to investigate the bifurcational patterns and the dynamical behavior of the reactor as a function of different key operating parameters. Suspended cells and biofilm are assumed to grow according to double limiting kinetics with phenol inhibition (carbon source) and oxygen limitation. The model presented by Russo et al. is extended to embody key features of the phenomenology of the granular‐supported biofilm: biofilm growth and detachment, gas–liquid oxygen transport, phenol, and oxygen uptake by both suspended and immobilized cells, and substrate diffusion into the biofilm. Steady‐state conditions and stability, and local dynamic behavior have been characterized. The multiplicity of steady states and their stability depend on key operating parameter values (dilution rate, gas–liquid mass transfer coefficient, biofilm detachment rate, and inlet substrate concentration). Small changes in the operating conditions may be coupled with a drastic change of the steady‐state scenario with transcritical and saddle‐node bifurcations. The relevance of concentration profiles establishing within the biofilm is also addressed. When the oxygen level in the liquid phase is <10% of the saturation level, the biofilm undergoes oxygen starvation and the active biofilm fraction becomes independent of the dilution rate. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Antiserum directed against cell surface antigens is lethal toLeishmania donovani promastigotes

The purified flagellar fraction ofLeishmania donovani promastigotes consists of 30–35 polypeptides. Antiserum raised against this fraction reacts with both flagella and pellicular membrane antigens as evident from immunoblot and immunofluorescence studies. Only 3 of these immunoreactive polypeptides are flagellum-specific. The antiserum agglutinates the cells and inhibits their growth in liquid culture medium. Moreover, glucose uptake and glucose-stimulated oxygen uptake of the promastigotes are significantly inhibited by the antiserum. The results indicate that the antiserum has a profound lethal effect on the invitro propagation of the parasite.     

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.     

Hydrogel-perfluorocarbon composite scaffold promotes oxygen transport to immobilized cells

Cell encapsulation provides cells a three-dimensional structure to mimic physiological conditions and improve cell signaling, proliferation, and tissue organization as compared to monolayer culture. Encapsulation devices often encounter poor mass transport, especially for oxygen, where critical dissolved levels must be met to ensure both cell survival and functionality. To enhance oxygen transport, we utilized perfluorocarbon (PFC) oxygen vectors, specifically perfluorooctyl bromide (PFOB) immobilized in an alginate matrix. Metabolic activity of HepG2 liver cells encapsulated in 1% alginate/10% PFOB composite system was 47-104% higher than alginate systems lacking PFOB. A cubic model was developed to understand the oxygen transport mechanism in the alginate/PFOB composite system. The theoretical flux enhancement in alginate systems containing 10% PFOB was 18% higher than in alginate-only systems. Oxygen uptake rates (OURs) of HepG2 cells were enhanced with 10% PFOB addition under both 20% and 5% O2 boundary conditions, by 8% and 15%, respectively. Model predictions were qualitatively and quantitatively verified with direct experimental OUR measurements using both a perfusion reactor and oxygen sensing plate, demonstrating a greater OUR enhancement under physiological O2 boundary conditions (i.e., 5% O2). Inclusion of PFCs in an encapsulation matrix is a useful strategy for overcoming oxygen limitations and ensuring cell viability and functionality both for large devices (>1 mm) and over extended time periods. Although our results specifically indicate positive enhancements in metabolic activity using the model HepG2 liver system encapsulated in alginate, PFCs could be useful for improving/stabilizing oxygen supply in a wide range of cell types and hydrogels.     

Immobilization of yeast cells by entrapment and adhesion using siliceous materials

Yeast cells (Saccharomyces cerevisiae) have been immobilized by entrapment in silica hydrogel, without significantly changing their biological activity; a simple model describes the rate of oxygen uptake by a film of immobilized cells. The cells have also been immobilized by direct adhesion to a glass surface; this is achieved by a well-controlled drying procedure, sufficient to bring the cells into close contact with the support, but without cell dehydration. The immobilized cells consume glucose at a rate which is about half of the rate obtained in suspension and they are resistant to strong mechanical strains.     

Determination of carbon dioxide evolution rate using on-line gas analysis during dynamic biodegradation experiments

Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (K(L)a) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.     

Respiratory inhibition by copper in Tetrahymena pyriformis GL

An excess of copper incorporated into Tetrahymena cells was mainly distributed in mitochondria, and inhibited oxygen uptake of Tetrahymena cells. The inhibition of oxygen uptake was clearly to copper uptake in mitochondria. Succinate was most favorable as a substrate stimulating oxygen uptake in mitochondria, and oxygen uptake was most strongly inhibited by copper (0.1 mM) in the presence of succinate among various substrates. The copper incorporated into mitochondria was in the fraction with the inner membranes. Succinate dehydrogenase (SDH) was inhibited at the lowest copper concentration (0.1 mM) among respiratory related enzymes. The redox potential of respiratory components was raised by copper. These results suggest that respiratory inhibition of Tetrahymena cells by copper may be mainly cause by inhibition of SDH as a FAD-protein and oxidation of electron carriers. At higher copper concentrations, MDH, cytochrome c reductase, and ATP synthesis were also inhibited. Growth inhibition may be due to these effects of copper in mitochondria. Mercury affected both oxygen uptake and SDH more strongly than copper. Zinc (0.1 mM) also affected oxygen uptake in mitochondria and a little in whole cells, however, it did not inhibit SDH. Cobalt, manganese, and nickel affected both oxygen uptake and SDH only a little at the same concentration (0.1 mM) as copper.     

Oxidative Activities of Mitochondria from Callus and Tumour Tissues3Rumex acetosa L.

The capacity of mitochondria from callus and virus tumour tissuecultures of Rumex acetosa L. to oxidize Krebs-cycle acids wasinvestigated. The rate of oxygen uptake of callus mitochondriawas considerably greater. The rate of substrate oxidation bymitochondria from both tissues decreased with age of the culture.Callus cells were found to contain considerably greater quantitiesof mitochondria than tumour cells. The oxidative activitiesof mitochondria and growth-rates of tissues were affected bythe composition of the culture medium. There was no direct relationshipbetween growth-rate and oxidative activity.     

New insights on O<Subscript>2</Subscript> uptake mechanisms in two-phase partitioning bioreactors

Numerous reports show that both transfer and uptake of poorly-water soluble substrates are significantly enhanced in two-phase partitioning bioreactors (TPPBs). A number of hypotheses have been put forward to explain these enhancements and among them, the occurrence of direct substrate or oxygen uptake from the vector/water interface has been suggested. The objective of this paper was to quantify the direct oxygen uptake from the vector/water interface in a culture of Pseudomonas putida, performed in a stirred tank reactor, using glucose as substrate and silicone oil as vector. Despite of a sufficient dissolved O₂ concentration in the vector phase (17 mg l⁻¹) and a significant vector surface area (4,000 m⁻¹) no significant direct O₂ uptake from the vector/water interface was observed, compared to O₂ uptake from the aqueous phase. From these results it was concluded that, direct O₂ or substrate uptake from the vector/water interface might not be significant in TPPBs.     

Stoichiometric model of the aerobic metabolism of the biological phosphorus removal process

In the aerobic phase of the biological phosphorus removal process, poly-beta-hydroxybutyrate, produced during anaerobic conditions, is used for cell growth, phosphate uptake, and glycogen formation. A metabolic model of this process has been developed. The yields for growth, polyphosphate and glycogen formation are quantified using the coupling of all these conversions to the oxygen consumption. The uptake of phosphate and storage as polyphosphate is shown to have a direct effect on the observed oxygen consumption in the aerobic phase. The overall energy requirements for the P-metabolism are substantial: 25% of the acetate consumed during anaerobic conditions and 60% of the oxygen consumptions is used for the synthesis of polyphosphate and glycogen. (c) 1994 John Wiley & Sons, Inc.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

CSF-responsive bone marrow cells in liquid culture: characterization and screening applications

In a previous study, colony-stimulating factor (CSF) activity assayed in colony culture correlated closely with 3HTdR uptake by human marrow cells depleted of adherent cells. To use this assay for screening media for CSF and immunotoxins for marrow toxicity, cells growing in liquid culture were compared to conventional granulocyte/macrophage (CFU-gm) colony assays. CSF dose-response relationships for liquid and colony-forming assays were nearly identical. 3HTdR uptake by nonadherent marrow cells was CSF dose-related, and there was a linear relationship between number of cells cultured and 3HTdR uptake. Ricin cytotoxicity curves for liquid cultures and CFU-gm were identical on day 7 but showed some disparity with day 14 cultures. Results with all cultures showed 3HTdR uptake to be most closely correlated with CFU-gm colony, rather than cluster, growth. Myeloid cell differentiation in liquid culture was similar to colony cultures, producing mixtures of granulocytes, macrophages and eosinophils. By combining cell and differential counts, production of various myeloid cells could be quantitated. Cytotoxicity of anti-Ia for CFU-gm and liquid culture cells was compared and the majority of both cell populations expressed Ia-like antigens. Simultaneous staining for surface antigens and DNA content was used to characterize proliferating marrow cells, and the vast majority of cells expressed myeloid markers. Transferrin receptors were displayed by cells in S/G2/M and appeared after CSF stimulation on G0/G1 cells. We conclude liquid cultures can be used to screen conditioned media for human CSF and to screen for cytotoxicity to normal myeloid precursor cells. Behavior of CSF-responsive cells in liquid culture appears most closely related to that of CFU-gm colony-forming cells, and characterization of CSF-stimulated cells allows quantitative as well as qualitative estimates of myeloid cell production.     

Some effects of light on algal respiration and the validity of the light and dark hottle technique for measuring primary productivity

SUMMARY. Measurements of the rate of oxygen uptake in a number of blue-green algae and diatoms were carried out under both field and laboratory conditions to determine the effects of light on such rates. The light history of algal cells was an important controlling factor of oxygen uptake. When measured in the light, with dichlorophenyl-dimethylurea (DCMU), oxygen uptake was sometimes different from uptake measured in the dark. The results cast some doubt on the validity of the light and dark bottle method for determining primary productivity. It is suggested that oxygen uptake measurements should be made in the presence of DCMU.     

Modelling and dynamic simulation of a moving bed bioreactor using respirometry for the estimation of kinetic parameters

Respirometry was used for the characterization of active autotrophic and heterotrophic biomass in a pilot scale moving bed bioreactor (MBBR). For this purpose biofilm samples attached to the carrier elements of the MBBR were transferred to a static gas/static liquid type respirometer with intermittent aeration. Known amounts of ammonia nitrogen and acetate were added to the respirometer. The dissolved oxygen (DO) decrease during the non-aeration phase was measured and used to calculate the oxygen uptake rate (OUR) of the active biomass. The resulting respirograms featured the typical endogenous and exogenous respiration phases and the shape of the respirogram was as expected from analogous respirometry with activated sludge. The OUR response was modelled with the activated sludge model ASM1 and the relevant kinetic parameter values for autotrophic and heterotrophic growth were adjusted. The adjusted parameter set and data originating from a 4-day long intensive measurement campaign were used for modelling and simulation of the pilot scale MBBR. The parameter estimation resulted in a good dynamic simulation of ammonia and nitrate variations in the effluent of the MBBR. Important MBBR properties including biofilm age, biofilm composition, and both attachment and detachment rate were extracted from the model.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.