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High Cell Density (high + cell_density)
Terms modified by High Cell Density Selected AbstractsDevelopment of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intronFEMS MICROBIOLOGY LETTERS, Issue 2 2010Jong Myoung Park Abstract Ralstonia eutropha H16 is a Gram-negative lithoautotrophic bacterium and is one of the best biopolymer-producing bacteria. It can grow to high cell densities either under lithoautotrophic or under heterotrophic conditions, which makes it suitable for a number of biotechnological applications. Also, R. eutropha H16 can degrade various aromatic compounds for environmental applications. The mobile group II intron can be used for the rapid and specific disruption of various bacterial genes by insertion into any desired target genes. Here, we applied the mobile group II intron to R. eutropha H16 and developed a markerless gene knockout system for R. eutropha: RalsTron. As a demonstration of the system, the phaC1 gene encoding polyhydroxyalkanoate synthase was successfully knocked out in R. eutropha H16. Furthermore, this knockout system would be useful for knocking out genes in other bacteria as well because it is based on a broad-host-range vector and the mobile group II intron that minimally depends on the bacterial hosts. [source] Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoterMOLECULAR MICROBIOLOGY, Issue 4 2002Gabriela Kovacikova Summary Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a ,hapR mutant and decreased in a ,luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between ,85 and ,58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at ,85 and ,77. Introducing the ,77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a ,luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes. [source] Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulenceMOLECULAR MICROBIOLOGY, Issue 2 2002Yaowei Kang Summary As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities. [source] Axenic Culture of the Heterotrophic Dinoflagellate Pfiesteria shumwayae in a Semi-Defined MediumTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 1 2009HAYLEY M. SKELTON ABSTRACT. A semi-defined, biphasic culture medium was developed that supported the axenic growth of three strains of the heterotrophic dinoflagellate Pfiesteria shumwayae. Maximum cell yields and division rates in the semi-defined medium ranged from 0.1 × 105 to 4.0 × 105 cells/ml and 0.5 to 1.7 divisions/day, respectively, and depended on the concentration of the major components in the medium as well as the P. shumwayae strain. The medium contained high concentrations of certain dissolved and particulate organic compounds, including amino acids and lipids. Pfiesteria shumwayae flagellated cells were attracted to insoluble lipids present in the medium and appeared to feed on the lipid particles, suggesting that phagocytosis may be required for growth in axenic culture. Development of a semi-defined medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of the biochemical requirements of P. shumwayae, needed to advance understanding of the nutritional ecology of this species. Further, this medium provides an economical, simplified method for generating high cell densities of P. shumwayae in axenic culture that will facilitate controlled investigations on the physiology and biochemistry of this heterotrophic dinoflagellate. [source] Process optimization of the integrated synthesis and secretion of ectoine and hydroxyectoine under hyper/hypo-osmotic stressBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010C. Fallet Abstract The synthesis and secretion of the industrial relevant compatible solutes ectoine and hydroxyectoine using the halophile bacterium Chromohalobacter salexigens were studied and optimized. For this purpose, a cascade of two continuously operated bioreactors was used. In the first bioreactor, cells were grown under constant hyperosmotic conditions and thermal stress driving the cells to accumulate large amounts of ectoines. To enhance the overall productivity, high cell densities up to 61,g,L,1 were achieved using a cross-flow ultrafiltration connected to the first bioreactor. In the coupled second bioreactor the concentrated cell broth was subjected to an osmotic and thermal down-shock by addition of fresh distilled water. Under these conditions, the cells are forced to secrete the accumulated intracellular ectoines into the medium to avoid bursting. The cultivation conditions in the first bioreactor were optimized with respect to growth temperature and medium salinity to reach the highest synthesis (productivity); the second bioreactor was optimized using a multi-objective approach to attain maximal ectoine secretion with simultaneous minimization of cell death and product dilution caused by the osmotic and thermal down-shock. Depending on the cultivation conditions, intracellular ectoine and hydroxyectoine contents up to 540 and 400,mg per g cell dry weight, respectively, were attained. With a maximum specific growth rate of 0.3,h,1 in defined medium, productivities of approximately 2.1,g,L,1,h,1 secreted ectoines in continuous operation were reached. Biotechnol. Bioeng. 2010;107: 124,133. © 2010 Wiley Periodicals, Inc. [source] Twenty-four-well plate miniature bioreactor high-throughput system: Assessment for microbial cultivationsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2007Kevin Isett Abstract High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (µ)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the µ-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6,±,2.4, 46.5,±,4.6, 51.6,±,3.7, and 56.1,±,1.6 h,1 at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively. Biotechnol. Bioeng. 2007;98: 1017,1028. © 2007 Wiley Periodicals, Inc. [source] Reassessing culture media and critical metabolites that affect adenovirus productionBIOTECHNOLOGY PROGRESS, Issue 1 2010Chun Fang Shen Abstract Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 × 106 cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 × 106 cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 × 106 cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Ferrous iron oxidation by foam immobilized Acidithiobacillus ferrooxidans: Experiments and modelingBIOTECHNOLOGY PROGRESS, Issue 5 2009S. Jaisankar Abstract Ferrous iron bio-oxidation by Acidithiobacillus ferrooxidans immobilized on polyurethane foam was investigated. Cells were immobilized on foams by placing them in a growth environment and fully bacterially activated polyurethane foams (BAPUFs) were prepared by serial subculturing in batches with partially bacterially activated foam (pBAPUFs). The dependence of foam density on cell immobilization process, the effect of pH and BAPUF loading on ferrous oxidation were studied to choose operating parameters for continuous operations. With an objective to have high cell densities both in foam and the liquid phase, pretreated foams of density 50 kg/m3 as cell support and ferrous oxidation at pH 1.5 to moderate the ferric precipitation were preferred. A novel basket-type bioreactor for continuous ferrous iron oxidation, which features a multiple effect of stirred tank in combination with recirculation, was designed and operated. The results were compared with that of a free cell and a sheet-type foam immobilized reactors. A fivefold increase in ferric iron productivity at 33.02 g/h/L of free volume in foam was achieved using basket-type bioreactor when compared to a free cell continuous system. A mathematical model for ferrous iron oxidation by Acidithiobacillus ferrooxidans cells immobilized on polyurethane foam was developed with cell growth in foam accounted by an effectiveness factor. The basic parameters of simulation were estimated using the experimental data on free cell growth as well as from cell attachment to foam under nongrowing conditions. The model predicted the phase of both oxidation of ferrous in shake flasks by pBAPUFs as well as by fully activated BAPUFs for different cell loadings in foam. Model for stirred tank basket bioreactor predicted within 5% both transient and steady state of the experiments closely for the simulated dilution rates. Bio-oxidation at high Fe2+ concentrations were simulated with experiments when substrate and product inhibition coefficients were factored into cell growth kinetics. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Cell density- and ComE-dependent expression of a group of mutacin and mutacin-like genes in Streptococcus mutansFEMS MICROBIOLOGY LETTERS, Issue 1 2006Jens Kreth Abstract Streptococcus mutans is a major cariogenic inhabitant of the high cell density oral biofilm (dental plaque). In previous studies, we showed that production of one of its virulence factors, the bacteriocin mutacin IV, was regulated by high cell density as well as the competence regulatory system ComED. In this study, we utilized luciferase fusions and real-time reverse transcriptase polymerase chain reaction (RT-PCR), to demonstrate that high cell density and ComED also regulate an uncharacterized group of mutacin and mutacin-like genes. Under high cell density or in the presence of externally added competence-stimulating peptide (CSP), gene expression increased 10- to 30-fold. Interestingly, high cell density was able to bypass the requirement for CSP addition. However, both cell density and CSP-dependent gene expression had a strict requirement for the ComE response regulator. [source] Role of phospholipases A2 in growth-dependent changes in prostaglandin release from 3T6 fibroblastsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2001Teresa Sánchez Previously, we reported a growth-dependent change in prostaglandin production as a consequence of a marked growth-dependent alteration in arachidonic acid (AA) mobilization from phospholipids. Our present results show that fetal calf serum (FCS) and 4,-phorbol-12-myristate acetate (PMA) caused an enhancement of phospholipase A2 (PLA2) activity in the membrane fraction of non-confluent cells allowing PLA2 access to its substrate and the release of AA. Western blot analysis has shown that FCS and PMA increased secreted PLA2 (sPLA2) expression in non-confluent 3T6 fibroblast cultures. Moreover, FCS and PMA induced dithiothreitol-sensitive and bromoenol lactone-sensitive PLA2 activities in cytosol and membrane fraction. However, these stimuli did not modify significantly the PLA2 activity in both fractions when 3T6 fibroblasts reached a high cell density. This could be associated with the impairment of AA mobilization in these cell culture conditions. On the other hand, we observed that FCS and PMA induced the same prostaglandin H synthase-2 induction in non-confluent and confluent culture conditions. Moreover, the prostaglandin E2 levels reached in cell culture supernatants were independent of the degree of confluence when AA was added exogenously. These results suggest that the changes of intracellular distribution of PLA2 activity of sPLA2 and iPLA2 stimulated by exogenous stimuli may be controlled by cell density conditions which constitute an important mechanism in the regulation of prostaglandin release.© 2001 Wiley-Liss, Inc. [source] Rhizoremediation of lindane by root-colonizing SphingomonasMICROBIAL BIOTECHNOLOGY, Issue 1 2008Dietmar Böltner Summary We used a two-step enrichment approach to isolate root-colonizing hexachlorocyclohexane (HCH)-degrading microorganisms. The first step consists of the use of classical liquid enrichment to isolate ,-HCH degraders. The ,-HCH-degrading microbes were attached in mass to corn seeds sown in soil with ,-HCH, and after plant development we rescued bacteria growing on root tips. Bacteria were then subjected to a second enrichment round in which growth on liquid medium with ,-HCH and inoculation of corn seeds were repeated. We then isolated bacteria on M9 minimal medium with ,-HCH from root tips. We were able to isolate four Sphingomonas strains, all of which degraded ,-, ,-, ,- and ,-HCH. Two of the strains were particularly good colonizers of corn roots, reaching high cell density in vegetated soil and partly removing ,-HCH. In contrast, these bacteria performed poorly in unplanted soils. This study supports the hypothesis that the removal of persistent toxic chemicals can be accelerated by combinations of plants and bacteria, a process generally known as rhizoremediation. [source] Quorum sensing controls biofilm formation in Vibrio choleraeMOLECULAR MICROBIOLOGY, Issue 1 2003Brian K. Hammer Summary Multiple quorum-sensing circuits function in parallel to control virulence and biofilm formation in Vibrio cholerae. In contrast to other bacterial pathogens that induce virulence factor production and/or biofilm formation at high cell density in the presence of quorum-sensing autoinducers, V. cholerae represses these behaviours at high cell density. Consistent with this, we show here that V. cholerae strains ,locked' in the regulatory state mimicking low cell density are enhanced for biofilm production whereas mutants ,locked' in the regulatory state mimicking high cell density are incapable of producing biofilms. The quorum-sensing cascade we have identified in V. cholerae regulates the transcription of genes involved in exopolysaccharide production (EPS), and variants that produce EPS and form biofilms arise at high frequency from non-EPS, non-biofilm producing strains. Our data show that spontaneous mutation of the transcriptional regulator hapR is responsible for this effect. Several toxigenic strains of V. cholerae possess a naturally occurring frameshift mutation in hapR. Thus, the distinct environments occupied by this aquatic pathogen presumably include niches where cell-cell communication is crucial, as well as ones where loss of quorum sensing via hapR mutation confers a selective advantage. Bacterial biofilms could represent a complex habitat where such differentiation occurs. [source] The kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of ,-lactamase synthesisMOLECULAR MICROBIOLOGY, Issue 6 2003Valérie Duval Summary To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformis,-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various ,-lactam antibiotics were studied. The second-order acylation rate constants k2/K, ranged from 0.0017 to more than 1 µM,1s,1 and the deacylation rate constants were lower than 4 × 10,5 s,1. These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described. In the light of these results, the kinetics of ,-lactamase induction in Bacillus licheniformis were re-examined. When starting with a rather high cell density, a good ,-lactamase substrate such as benzylpenicillin is too sensitive to ,-lactamase-mediated hydrolysis to allow full induction. By contrast, a poor ,-lactamase substrate (7-aminocephalosporanic acid) can fully derepress ,-lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction. [source] Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulenceMOLECULAR MICROBIOLOGY, Issue 2 2002Yaowei Kang Summary As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities. [source] In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteriaMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 11 2007Robert E. Ward Abstract This study was conducted to investigate the catabolism and fermentation of human milk oligosaccharides (HMO) by individual strains of bifidobacteria. Oligosaccharides were isolated from a pooled sample of human milk using solid-phase extraction, and then added to a growth medium as the sole source of fermentable carbohydrate. Of five strains of bifidobacteria tested (Bifidobacterium longum biovar infantis, Bifidobacterium bifidum, Bifidobacterium longum biovar longum, Bifidobacterium breve, and Bifidobacterium adolescentis), B. longum bv. infantis grew better, achieving triple the cell density then the other strains. B. bifidum did not reach a high cell density, yet generated free sialic acid, fucose and N-acetylglucosamine in the media, suggesting some capacity for HMO degradation. Thin layer chromatography profiles of spent fermentation broth suggests substantial degradation of oligosaccharides by B. longum bv. infantis, moderate degradation by B. bifidum and little degradation by other strains. While all strains were able to individually ferment two monosaccharide constituents of HMO, glucose and galactose, only B. longum bv. infantis and B. breve were able to ferment glucosamine, fucose and sialic acid. These results suggest that as a potential prebiotic, HMO may selectively promote the growth of certain bifidobacteria strains, and their catabolism may result in free monosaccharides in the colonic lumen. [source] Regional specialization of the Ganglion cell density in the retina of the Ostrich (Struthio camelus)ANIMAL SCIENCE JOURNAL, Issue 1 2010Mohammad L. RAHMAN ABSTRACT In this study, retinal whole-mount specimens were prepared and stained with 0.1% cresyl violet for the ganglion cell study in the Ostrich (Struthio camelus). The total number, distribution, and size of these cells were determined in different retinal regions. The mean total number of ganglion cells (three retinas) was 1 435 052 with an average density of 652 cells/mm2. The temporo , nasal area of the retina with high cell density were identified with the peak of 7525 cells/mm2 in the central area. The size of most ganglion cells ranged from 113,403 µm2, with smaller cells predominating along the temporo-nasal streak above the optic disc and larger cells comprising more of the peripheral regions. The average thickness of the retina was 196 µm. The central area was the thickest area (268.6 µm), whereas the peripheral area was the thinnest area. Thus, the specialization of ganglion cell densities, their sizes and the thickness of the retina support the notion that the conduction of visual information towards the brain from all regions of the retina is not uniform, and suggests that the temporo , nasal streak is the fine quality area for vision in ostriches. [source] NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2008Ying Zhu Abstract Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day,1 with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be ,30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing. Biotechnol. Bioeng. 2008;101: 751,760. © 2008 Wiley Periodicals, Inc. [source] Exploration of the hydrogen producing potential of Rhodobacter capsulatus chemostat cultures: The application of deceleration-stat and gradient-stat methodologyBIOTECHNOLOGY PROGRESS, Issue 5 2009Sebastiaan Hoekema Abstract In this work, the dependency of the volumetric hydrogen production rate of ammonium-limited Rhodobacter capsulatus chemostat cultures on their imposed biomass concentration and dilution rate was investigated. A deceleration-stat experiment was performed by lowering the dilution rate from 1.0 d,1 to zero aimed at a constant biomass concentration of 4.0 g L,1 at constant incident light intensity. The results displayed a maximal volumetric hydrogen production rate of 0.6 mmol m,3 s,1, well below model predictions. Possibly the high cell density limited the average light availability, resulting in a sub-optimal specific hydrogen production rate. To investigate this hypothesis, a gradient-stat experiment was conducted at constant dilution rate of 0.4 d,1 at constant incident light intensity. The biomass concentration was increased from 0.7 to 4.0 g L,1 by increasing the influent ammonium concentration. Up to a biomass concentration of 1.5 g L,1, the volumetric hydrogen production rate of the system increased according to model predictions, after which it started to decline. The results obtained provide strong evidence that the observed decline in volumetric hydrogen production rate at higher biomass concentrations was at least partly caused by a decrease in light availability. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Optimization of the Human Adenosine A2a Receptor Yields in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 5 2006Alison Wedekind G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A2a receptor (A2aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A2aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD600) and per cell A2aR expression levels. For these studies, the A2aR expression levels were determined using a previously described A2aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times (,60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A2aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points (,60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization. [source] Effects of Oxygen Transport on 3-D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures: Experiments and Mathematical ModelBIOTECHNOLOGY PROGRESS, Issue 4 2005Feng 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] Production of a Secreted Glycoprotein from an Inducible Promoter System in a Perfusion BioreactorBIOTECHNOLOGY PROGRESS, Issue 5 2004Matthew L. Lipscomb The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated. [source] Optimization of Cyclodextrin Glycosyltransferase Production from KlebsiellapneumoniaeAS-22 in Batch, Fed-Batch, and Continuous CulturesBIOTECHNOLOGY PROGRESS, Issue 6 2003Bharat N. Gawande Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniaeAS-22 strain, which converts starch predominantly to ,-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media. [source] | ||||||||||||||||||||||