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Oxygen Limitation (oxygen + limitation)

Distribution by Scientific Domains

Life Sciences	83%

Selected Abstracts

Evaluation of a critical process parameter: Oxygen limitation during cultivation has a fully reversible effect on gene expression of Bordetella pertussis

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009
Mathieu Streefland
Abstract Modern (bio)pharmaceutical process development requires thorough investigation of all process parameters that are critical to product quality. The impact of a disturbance of such a parameter during processing needs to be known so that a rational decision can be made about the release of the product. In cultivation processes the dissolved oxygen (DO) concentration is generally accepted as being a critical parameter. In this article the impact of a 90 min period of oxygen limitation during the cultivation of the strictly aerobic Bordetella pertussis bacterium is investigated. The cultivation is the most important process step for the manufacturing of a vaccine against whooping cough disease. Samples were taken immediately before and after oxygen limitation and at the end of cultivation of four oxygen limited and three control cultivations. DNA microarray analysis of the full transcriptome of the B. pertussis bacterium revealed that a 90 min period of oxygen limitation has a substantial effect on overall gene expression patterns. In total 104 genes were identified as a significant hit at any of the sample points, of which 58 were directly related to oxygen limitation. The other genes were mainly affected towards the end of cultivation. Of all genes involved in oxygen limitation none were identified to show a significant difference between the oxygen limited and control cultivations at the end of the batch. This indicates a fully reversible effect of oxygen limitation on gene expression. This finding has implications for the risk assessment of dissolved oxygen concentration as a critical process parameter. Biotechnol. Bioeng. 2009;102: 161,167. © 2008 Wiley Periodicals, Inc. [source]

Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuni

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2010
Edward Guccione
Summary Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ,-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni -specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment. [source]

In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm

ENVIRONMENTAL MICROBIOLOGY, Issue 9 2005
Armin Gieseke
Summary Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e, equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers. [source]

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

FEMS YEAST RESEARCH, Issue 3 2003
C.Fredrik Wahlbom
Abstract The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding d -xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on d -xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only d -xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h,1 for S. cerevisiae TMB 3399 to 0.14 h,1 for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h,1. All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose,1 and 0.001, 0.10, and 0.16 g ethanol g biomass,1 h,1 for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein,1, was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant. [source]

Active brood care in Cancer setosus (Crustacea: Decapoda): the relationship between female behaviour, embryo oxygen consumption and the cost of brooding

FUNCTIONAL ECOLOGY, Issue 2 2002
J. A. Baeza
Summary 1,Previous studies have shown that oxygen is limiting in embryo masses of marine invertebrates. It has been suggested that several behaviours found in brooding females of brachyuran crabs are used to ventilate and provide oxygen to the embryo masses. 2,The relationship between female brooding behaviour, oxygen consumption of embryos and oxygen provision to the brood mass for embryos at different developmental stages was studied, using the marine crab Cancer setosus. The changes in oxygen consumption of brooding females associated with changes in oxygen provision to the brood were also estimated. 3Brooding females of C. setosus behaved differently from non-brooding females. Abdominal flapping was associated with an increase in oxygen availability in the centre of the brood mass; the frequency of abdominal flapping increased with embryonic development, as oxygen demand of crab embryos increased. Oxygen consumption of brooding females also increased throughout embryonic development. The difference in oxygen consumption between brooding and non-brooding females was used as an indicator of the cost of oxygen provision (brooding). 4,These results provide the first evidence , among crabs and other marine invertebrates , of a direct link between active brood care and oxygen provision. It is possible that parental care in marine invertebrates is strongly linked to oxygen provision, since oxygen limitation has been reported for several brooding taxa. The simple physiological constraint of oxygen provision in marine invertebrates may have important ecological and evolutionary consequences. [source]

Optimization of C:N ratio and minimal initial carbon source for poly(3-hydroxybutyrate) production by Bacillus megaterium

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 12 2009
Débora Jung Luvizetto Faccin
Abstract BACKGROUND: The aim of this research was the optimization of poly(3-hydroxybutyrate),P(3HB),production in submerged cultures of Bacillus megaterium in a mineral medium using sucrose as carbon source and nitrogen as the limiting substrate. Small-scale experiments were carried out in shake flasks at 30 °C and 160 rpm in order to evaluate the best initial sucrose concentration and carbon:nitrogen ratio to maximize biomass accumulation and biopolymer production. An objective function in terms of residual sucrose and P(3HB) production was proposed in order to optimize the amount of carbon source used and the production of P(3HB). RESULTS: High production of P(3HB) was obtained, with approximately 70% (CDW) accumulation in cells without nitrogen limitation and strongly correlated with the pH of the culture. Scaling-up the system to cultures in a bioreactor, with or without pH control, a reduction of P(3HB) accumulation (around 30% CDW) was observed when compared with shaker cultures, suggesting a possible role of oxygen limitation as a stress signaling for P(3HB) synthesis. CONCLUSIONS: Results of our experiments showed that Bacillus megaterium was able to produce P(3HB) at one of the highest production rates so far reported for this bacterium, making this microorganism very interesting for industrial applications. Comparisons of shaker and bench-scale bioreactor experiments show both the importance of pH and aeration strategies. It is likely that complex aeration strategies linked to cell metabolism will be necessary for further developments using this bacterium. Copyright © 2009 Society of Chemical Industry [source]

Mathematical modeling of gas-phase biofilter performance

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2003
Hasnaa Jorio
Abstract In the present paper, a new mathematical model describing the physical, chemical and biological phenomena involved in the process of contaminant removal in biofilters is developed. In addition to the contaminant, the key components of the present theoretical model are carbon dioxide and oxygen. The model predicts the concentration profile of the key components in the gas phase, the biofilm and the sorption liquid retained in the solid particles composing the filter bed at both steady and transient regimes. The model equations were solved numerically and comparison between theory and experiment showed that the model results for styrene and carbon dioxide concentration profiles were in very good agreement with experimental data for the biofiltration of styrene vapors at steady state. The analysis of oxygen concentration profile in the biofilm predicted by the theoretical model revealed that oxygen limitation does not occur under the operating styrene biodegradation rate in the biofilter. Copyright © 2003 Society of Chemical Industry [source]

Temperature-dependent changes in energy metabolism, intracellular pH and blood oxygen tension in the Atlantic cod

JOURNAL OF FISH BIOLOGY, Issue 6 2003
F. J. Sartoris
The effect of acute increase in temperature on oxygen partial pressure (Po2) was measured in the gill arches of Atlantic cod Gadus morhua between 10 and 19° C by use of oxygen microoptodes. Oxygen saturation of the gill blood under control conditions varied between 90 and 15% reflecting a variable percentage of arterial or venous blood in accordance with the position of each optode in the gill arch. The data obtained suggested that arterial Po2 remained more or less constant and arterial oxygen uptake did not become limiting during warming. A progressive drop in venous Po2, however, was observed at >10° C indicating that excessive oxygen uptake from the blood is not fully compensated for by circulatory performance, until finally, Po2 levels fully collapse. In a second set of experiments energy and acid,base status of white muscle of Atlantic cod in vivo was measured by magnetic resonance (31P-NMR) spectroscopy in unanaesthetized and unimmobilized fish in the temperature range between 13 and 21° C. A decrease in white muscle intracellular pH (pHi) with temperature occurred between 10 and 16° C (,pH per ° C = ,0·025 per ° C). In white muscle temperature changes had no influence on high-energy phosphates such as phosphocreatine (PCr) or ATP except during exposure to high critical temperatures (>16° C), indicating that white muscle energy status appears to be relatively insensitive to thermal stress if compared to the thermal sensitivity of the whole animal. The data were consistent with the hypothesis of an oxygen limitation of thermal tolerance in animals, which is set by limited capacity of oxygen supply mechanisms. In the case of Atlantic cod circulatory rather than ventilatory performance may be the first process to cause oxygen deficiency during heat stress. [source]

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses,

BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009
Alvaro R. Lara
Abstract The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed-acid fermentation metabolism was studied in a two-compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini-plug-flow reactor (PFR: BioScope, 3.5,mL volume). Such a system can mimic the situation often encountered in large-scale, fed-batch bioreactors. The STR represented the zones of a large-scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen-poor and glucose-rich regions can occur in large-scale bioreactors. The response of E. coli to these large-scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D,=,0.1,h,1) into the mini-PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92,s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2,s to the glucose-rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2,s after of exposure of E. coli cells to the glucose and O2 gradients. This approach (STR-mini-PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large-scale bioreactors and for the design of modified strains with an improved behavior under large-scale conditions. Biotechnol. Bioeng. 2009; 104: 1153,1161. © 2009 Wiley Periodicals, Inc. [source]

Evaluation of a critical process parameter: Oxygen limitation during cultivation has a fully reversible effect on gene expression of Bordetella pertussis

Optimal fed-batch cultivation when mass transfer becomes limiting

BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007
John Villadsen
Abstract In the design of an aerobic fed-batch process to produce, for example, a pharmaceutical protein, the volumetric production rate will eventually become limited by mass transfer when the biomass concentration exceeds a certain upper limit x*. It appears to be common practice to switch from exponential feed of substrate to a constant feed rate when x* is reached. This is done to avoid oxygen starvation with a potential risk of undesired stress responses. But with a constant feed rate the carbon source (glucose) concentration may decrease to a low level with a resulting loss of viability and an undesired production of endotoxins. It is shown that an exponential feeding strategy may be continued, but with a smaller exponent than the one used before oxygen limitation occurs. This will diminish the potential detrimental effects on the culture due to low glucose concentration, and the total time to reach a given final biomass concentration will be reduced. Biotechnol. Bioeng. 2007;98: 706,710. © 2007 Wiley Periodicals, Inc. [source]

Noninvasive measurement of dissolved oxygen in shake flasks

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002
Leah Tolosa
Abstract Shake flasks are ubiquitous in cell culture and fermentation. However, conventional devices for measuring oxygen concentrations are impractical in these systems. Thus, there is no definitive information on the oxygen supply of growing cells. Here we report the noninvasive, nonintrusive monitoring of dissolved oxygen (DO) in shake flasks using a low-cost optical sensor. The oxygen-sensitive element is a thin, luminescent patch affixed to the inside bottom of the flask. The sensitivity and accuracy of this device is maximal up to 60% DO, within the range that is critical to cell culture applications. By measuring actual oxygen levels every 1 or 5 min throughout the course of yeast and E. coli fermentations, we found that a modest increase in shaker speed and a decrease in culture volume slowed the onset of oxygen limitation and reduced its duration. This is the first time that in situ oxygen limitation is reported in shake flasks. The same data is unattainable with a Clark type electrode because the presence of the intrusive probe itself changes the actual conditions. Available fiber optic oxygen sensors require cumbersome external connections and recalibration when autoclaved. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 594,597, 2002. [source]

Effects of Oxygen Transport on 3-D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures: Experiments and Mathematical Model

BIOTECHNOLOGY PROGRESS, Issue 4 2005
Feng Zhao
Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 ,mol/106 cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher YL/G in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering [source]

Xylitol Production from Sugarcane Bagasse Hydrolyzate in Fluidized Bed Reactor.

BIOTECHNOLOGY PROGRESS, Issue 4 2003
Effect of Air Flowrate
Cells of Candida guilliermondiiimmobilized onto porous glass spheres were cultured batchwise in a fluidized bed bioreactor for xylitol production from sugarcane bagasse hemicellulose hydrolyzate. An aeration rate of only 25 mL/min ensured minimum yields of xylose consumption (0.60) and biomass production (0.14 gDM/gXyl), as well as maximum xylitol yield (0.54 gXyt/gXyl) and ratio of immobilized to total cells (0.83). These results suggest that cell metabolism, although slow because of oxygen limitation, was mainly addressed to xylitol production. A progressive increase in the aeration rate up to 140 mL/min accelerated both xylose consumption (from 0.36 to 0.78 gXyl/L·h) and xylitol formation (from 0.19 to 0.28 gXyt/L·h) but caused the fraction of immobilized to total cells and the xylitol yield to decrease up to 0.22 and 0.36 gXyt/gXyl, respectively. The highest xylitol concentration (17.0 gXyt/L) was obtained at 70 mL/min, but the specific xylitol productivity and the xylitol yield were 43% and 22% lower than the corresponding values obtained at the lowest air flowrate, respectively. The concentrations of consumed substrates and formed products were used in material balances to evaluate the xylose fractions consumed by C. guilliermondii for xylitol production, complete oxidation through the hexose monophosphate shunt, and cell growth. The experimental data collected at variable oxygen level allowed estimating a P/O ratio of 1.35 molATP/molO and overall ATP requirements for biomass growth and maintenance of 3.4 molATP/C-molDM. [source]

Novel Hemoglobins to Enhance Microaerobic Growth and Substrate Utilization in Escherichiacoli,

BIOTECHNOLOGY PROGRESS, Issue 5 2001
Christian J. T. Bollinger
Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiellapneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmpgenes from P. aeruginosa, S. typhi, C. jejuni, K.pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A600 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A600 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D.radiodurans hemoprotein-expressing E. colirelative to the E. coli control carrying the parental plasmid without any hemoglobin gene. [source]

Hydrogel-Perfluorocarbon Composite Scaffold Promotes Oxygen Transport to Immobilized Cells

BIOTECHNOLOGY PROGRESS, Issue 2 2008
Kyuongsik Chin
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. [source]

Hairy Root Culture in a Liquid-Dispersed Bioreactor: Characterization of Spatial Heterogeneity

BIOTECHNOLOGY PROGRESS, Issue 3 2000
Gary R. C. Williams
A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7. Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity developed in the vessel. Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor. Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor. Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed. Roots near the bottom of the vessel trapped up to 3,4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems. Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed. Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance. The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance. Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems. [source]