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Continuous Culture (continuous + culture)
Selected AbstractsUNCOUPLING OF SILICON COMPARED WITH CARBON AND NITROGEN METABOLISMS AND THE ROLE OF THE CELL CYCLE IN CONTINUOUS CULTURES OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) UNDER LIGHT, NITROGEN, AND PHOSPHORUS CONTROL1JOURNAL OF PHYCOLOGY, Issue 5 2002Pascal Claquin The elemental composition and the cell cycle stages of the marine diatom Thalassiosira pseudonana Hasle and Heimdal were studied in continuous cultures over a range of different light- (E), nitrogen- (N), and phosphorus- (P) limited growth rates. In all growth conditions investigated, the decrease in the growth rate was linked with a higher relative contribution of the G2+M phase. The other phases of the cell cycle, G1 and S, showed different patterns, depending on the type of limitation. All experiments showed a highly significant increase in the amount of biogenic silica per cell and per cell surface with decreasing growth rates. At low growth rates, the G2+M elongation allowed an increase of the silicification of the cells. This pattern could be explained by the major uptake of silicon during the G2+M phase and by the independence of this process on the requirements of the other elements. This was illustrated by the elemental ratios Si/C and Si/N that increased from 2- to 6-fold, depending of the type of limitation, whereas the C/N ratio decreased by 10% (E limitation) or increased by 50% (P limitation). The variations of the ratios clearly demonstrate the uncoupling of the Si metabolism compared with the C and N metabolisms. This uncoupling enabled us to explain that in any of the growth condition investigated, the silicification of the cells increased at low growth rates, whereas carbon and nitrogen cellular content are differently regulated, depending of the growth conditions. [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] Modeling Stability of Photoheterotrophic Continuous Cultures in PhotobioreactorsBIOTECHNOLOGY PROGRESS, Issue 4 2003Jean-François Cornet Continuous cultures of the purple non-sulfur bacterium Rhodospirillum rubrum were grown in a cylindrical photobioreactor in photoheterotrophic conditions, using acetate as carbon source. A new kinetic and stoichiometric knowledge model was developed, and its ability to simulate experimental results obtained under varying incident light fluxes and residence times is discussed. The model accurately predicts the stable, unstable, or oscillating behavior observed for the reactor productivity. In particular, the values of residence time corresponding to a subcritical bifurcation with a typical hysteresis effect are calculated and analyzed. The robustness of the proposed model allows the engineering operating domain of the photobioreactor function to be set and offers a promising tool for the design and control of such photoheterotrophic processes. [source] Cell Population Modeling and Parameter Estimation for Continuous Cultures of Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 5 2002Prashant Mhaskar Saccharomyces cerevisiae is known to exhibit sustained oscillations in chemostats operated under aerobic and glucose-limited growth conditions. The oscillations are reflected both in intracellular and extracellular measurements. Our recent work has shown that unstructured cell population balance models are capable of generating sustained oscillations over an experimentally meaningful range of dilution rates. A disadvantage of such unstructured models is that they lack variables that can be compared directly to easily measured extracellular variables. Thus far, most of our work in model development has been aimed at achieving qualitative agreement with experimental data. In this paper, a segregated model with a simple structured description of the extracellular environment is developed and evaluated. The model accounts for the three most important metabolic pathways involved in cell growth with glucose substrate. As compared to completely unstructured models, the major advantage of the proposed model is that predictions of extracellular variables can be compared directly to experimental data. Consequently, the model structure is well suited for the application of estimation techniques aimed at determining unknown model parameters from available extracellular measurements. A steady-state parameter selection method developed in our group is extended to oscillatory dynamics to determine the parameters that can be estimated most reliably. The chosen parameters are estimated by solving a nonlinear programming problem formulated to minimize the difference between predictions and measurements of the extracellular variables. The efficiency of the parameter estimation scheme is demonstrated using simulated and experimental data. [source] A new photobioreactor for continuous microalgal production in hatcheries based on external-loop airlift and swirling flowBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Karine Loubière Abstract This study deals with the scale of a new photobioreactor for continuous microalgal production in hatcheries. The combination of the state-of-art with the constraints inherent to hatcheries has turned the design into a closed, artificially illuminated and external-loop airlift configuration based on a succession of elementary modules, each one being composed of two transparent vertical interconnected columns. The liquid circulation is ensured pneumatically (air injections) with respect to a swirling motion (tangential inlets). A single module of the whole photobioreactor was built-up to scale its geometry (diameter and length) and to optimize its design (air sparger, tangential inlets). The volumetric productivities were predicted by modeling radiative transfer and growth of Isochrysis affinis galbana (clone Tahiti). The hydrodynamics of the liquid phase was modeled in terms of global flow behavior (circulation and mixing times, Péclet number) and of swirling motion decay along the column (Particle Image Velocimetry). The aeration performances were determined by overall volumetric mass transfer measurements. Continuous cultures of Isochrysis affinis galbana (clone Tahiti) were run in two geometrical configurations, generating either an axial or a swirling flow. Lastly, the definitive options of design are presented as well as a 120-L prototype, currently implemented in a French mollusk hatchery and commercialized. Biotechnol. Bioeng. 2009;102: 132,147. © 2008 Wiley Periodicals, Inc. [source] Modeling Stability of Photoheterotrophic Continuous Cultures in PhotobioreactorsBIOTECHNOLOGY PROGRESS, Issue 4 2003Jean-François Cornet Continuous cultures of the purple non-sulfur bacterium Rhodospirillum rubrum were grown in a cylindrical photobioreactor in photoheterotrophic conditions, using acetate as carbon source. A new kinetic and stoichiometric knowledge model was developed, and its ability to simulate experimental results obtained under varying incident light fluxes and residence times is discussed. The model accurately predicts the stable, unstable, or oscillating behavior observed for the reactor productivity. In particular, the values of residence time corresponding to a subcritical bifurcation with a typical hysteresis effect are calculated and analyzed. The robustness of the proposed model allows the engineering operating domain of the photobioreactor function to be set and offers a promising tool for the design and control of such photoheterotrophic processes. [source] Dynamics of Benzene and Toluene Degradation in Pseudomonas putida F1 in the Presence of the Alternative Substrate SuccinateENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007I. Rüegg Abstract In batch and continuous culture, the regulation of benzene and toluene degradation by Pseudomonas putida,F1 was investigated in the presence of the alternative carbon and energy source succinate. In batch culture, benzene and toluene were used simultaneously, whereas succinate suppressed benzene consumption under carbon excess conditions resulting in diauxic growth. In carbon-limited continuous culture mixed substrate growth was observed. Since in nature low substrate concentrations and ever changing conditions prevail, this paper focuses on the dynamics of benzene/toluene degradation, biomass synthesis, and the regulation of benzene/toluene-degrading enzymes in cultures growing continuously at a dilution rate of 0.1,h,1, when shifting the supply of the carbon and energy source from succinate to various mixtures of succinate and benzene/toluene, or to benzene only. When the succinate concentration was kept constant (1.25,mM) and the medium was supplemented with benzene (2,mM), growth with benzene began already two hours after the shift. In contrast, replacing succinate with benzene only led to a wash out of biomass for more then ten hours, before biomass production from benzene started. A striking and reproducible transition pattern was observed for all shifts where the succinate concentration was reduced or succinate was omitted. After an initial period of biomass production from benzene, the culture collapsed and a wash-out of biomass was observed. However, this wash-out was not accompanied by an increase in benzene in the cultivation liquid, indicating a benzene uptake without conversion into biomass. Another possibility is that in phases of low biomass concentrations, cells were only able to use the low amounts of benzene/toluene dissolved in the cultivation liquid yielding low biomass concentrations whereas in phases of high biomass concentrations, they were able to rapidly utilize the aromatic solvents so that additional benzene from the gas phase diffused into the cultivation liquid resulting in more biomass production. In most cases, growth resumed again after 10 to 80,h. Currently, the reasons for the decrease in biomass after the first rise are unknown. However, several indications rule out intoxication of the cells by either the solvents benzene or toluene themselves, or by toxic degradation intermediates, or by-products. [source] Differential expression of antenna and core genes in Prochlorococcus PCC 9511 (Oxyphotobacteria) grown under a modulated light,dark cycleENVIRONMENTAL MICROBIOLOGY, Issue 3 2001Laurence Garczarek The continuous changes in incident solar light occurring during the day oblige oxyphototrophs, such as the marine prokaryote Prochlorococcus, to modulate the synthesis and degradation rates of their photosynthetic components finely. How this natural phenomenon influences the diel expression of photosynthetic genes has never been studied in this ecologically important oxyphotobacterium. Here, the high light-adapted strain Prochlorococcus sp. PCC 9511 was grown in large-volume continuous culture under a modulated 12 h,12 h light,dark cycle mimicking the conditions found in the upper layer of equatorial oceans. The pcbA gene encoding the major light-harvesting complex showed strong diel variations in transcript levels with two maxima, one before the onset of illumination and the other near the end of the photoperiod. In contrast, the mRNA level of psbA (encoding the reaction centre II subunit D1), the monocistronic transcript of psbD (encoding D2) and the dicistronic transcript of psbDC were all tightly correlated with light irradiance, with a minimum at night and a maximum at noon. The occurrence of a second peak during the dark period for the monocistronic transcript of psbC (encoding one of the PS II core Chl a antenna proteins) suggested the involvement of post-transcriptional regulation. Differential expression of the external antenna and core genes may constitute a mechanism of regulation of the antenna size to cope with the excess photon fluxes that Prochlorococcus cells experience in the upper layer of oceans around midday. The 5, ends of all transcripts were mapped, and a conserved motif, 5,-TTGATGA-3,, was identified within the putative psbA and pcbA promoters. [source] Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulphate and tetrachloroetheneENVIRONMENTAL MICROBIOLOGY, Issue 2 2001Oliver Drzyzga A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a ,biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium. [source] Bacterial competition between a bacteriocin-producing and a bacteriocin-negative strain of Streptococcus bovis in batch and continuous cultureFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Bruno M. Xavier Abstract A bacteriocin-producing Streptococcus bovis strain (HC5) outcompeted a sensitive strain (JB1) before it reached stationary phase (pH 6.4), even though it grew 10% slower and cell-free bovicin HC5 could not yet be detected. The success of bacteriocin-negative S. bovis isolates was enhanced by the presence of another sensitive bacterium (Clostridium sticklandii SR). PCR based on repetitive DNA sequences indicated that S. bovis HC5 was not simply transferring bacteriocin genes to S. bovis JB1. When the two S. bovis strains were coinoculated into minimal medium, bacteriocin-negative isolates predominated, and this effect could be explained by the longer lag time (0.5 vs. 1.5 h) of S. bovis HC5. If the glucose concentration of the minimal medium was increased from 2 to 7 mg mL,1, the effect of lag time was diminished and bacteriocin-producing isolates once again dominated the coculture. When the competition was examined in continuous culture, it became apparent that batch culture inocula were never able to displace a strain that had already reached steady state, even if the inoculum was large. This result indicated that bacterial selection for substrate affinity was even more important than bacteriocin production. [source] Order of genetic events is critical determinant of aberrations in chromosome count and structureGENES, CHROMOSOMES AND CANCER, Issue 4 2004Christine Fauth A sequential acquisition of genetic events is critical in tumorigenesis. A key step is the attainment of infinite proliferative potential. Acquisition of this immortalization requires the activation of telomerase in addition to other activities, including inactivation of TP53 and the retinoblastoma family of tumor-suppressor proteins. However, the importance of the order in which these genetic events occur has not been established. To address this question, we used a panel of normal mammary fibroblasts and endothelial cultures that were immortalized after transduction with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive mutant of the SV40 large-tumor (tsLT) oncoprotein in different orders in early- and late-passage stocks. These lines were maintained in continuous culture for up to 90 passages, equivalent to >300 population doublings (PDs) post-explantation during 3 years of continuous propagation. We karyotyped the cultures at different passages. Cultures that received hTERT first followed by tsLT maintained a near-diploid karyotype for more than 150 PDs. However, in late-passage stocks (>200 PDs), metaphase cells were mostly aneuploid. In contrast, the reverse order of gene transduction resulted in a marked early aneuploidy and chromosomal instability, already visible after 50 PDs. These results suggest that the order of genetic mutations is a critical determinant of chromosome count and structural aberration events. © 2004 Wiley-Liss, Inc. [source] Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperaturesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004E. van Rensburg Abstract Aims:, To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. Methods and Results:,Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, ,18 and ,75°C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain,heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. Conclusions:,Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. Significance and Impact of the Study:, Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen. [source] Aerobic biodegradation of MtBE in an upflow fixed bed reactorJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 6 2009Emma Bianchi Abstract BACKGROUND: An aerobic upflow fixed bed reactor (UFBR) was densely colonized by a bacterial consortium, obtained from gasoline polluted waters, able to mineralize MtBE and BTEX. The system was studied in order to determine its capability to degrade the MtBE present in prepared solutions and in real contaminated aquifers and was operating for more than a year. RESULTS: Efficient colonization of the reactor took about 50 days, utilizing bacteria grown in continuous culture in a fermenter connected to the UFBR. During the study the influence of feed concentration of MtBE, temperature and hydraulic retention time (HRT) was analyzed. The system, running at 18 °C on synthetic medium, was fed at an influent MtBE concentration of 27.8 mg L,1 with HRT of 5 h showing 99.98% of MtBE degradation. When working with polluted groundwater, the system achieved 100% BTEX degradation and 99.34% MtBE degradation. CONCLUSION: The UFBR was tested on synthetic medium spiked with MtBE and on groundwater contaminated with MtBE and BTEX at concentrations of 50,60 ppm and a few ppm, respectively. The reactor responded efficiently showing great flexibility and capability of adjustment to different operating conditions with MtBE degradation of nearly 100%. Copyright © 2009 Society of Chemical Industry [source] Ethanol production from raw starch by a recombinant yeast having saccharification and fermentation activitiesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2002Yoshitoshi Nakamura Abstract In order to develop a method for converting raw starch into ethanol efficiently, direct fermentation of ozonized raw starch using a recombinant yeast was investigated. Ozonolysis was carried out as a pretreatment to convert raw starch into ethanol rapidly and efficiently, and then the effect of the ozone degradation conditions on the degree of polymerization and the amount of amylose in a raw starch was determined. Since the degree of polymerization was low and the amount of amylose was high, raw starch treated with an ozone concentration of 40,gm,3 and an ozonation time of 30,min was the material chosen for alcohol fermentation. Though the recombinant yeast could not convert the untreated raw starch, it converted the soluble starch and the ozonized raw starch at a comparatively high yield into ethanol. About 56% of the ozonized raw starch decomposed, and the ethanol concentration obtained from the ozonized raw starch was markedly greater than that obtained from untreated raw starch. The dynamic behavior of cell growth, substrate degradation, and ethanol production was examined in a continuous culture under various dilution rates, and the optimal dilution rate, ie 0.15,h,1, was determined for maximizing the ethanol productivity (amount of ethanol produced per unit time). © 2002 Society of Chemical Industry [source] A structured model for the simulation of bioreactors under transient conditionsAICHE JOURNAL, Issue 11 2009Jérôme Morchain Abstract Modeling the transient behavior of continuous culture is of primary importance for the scale-up of biological processes. Spatial heterogeneities increase with the reactor size and micro-organisms have to cope with a fluctuating environment along their trajectories within the bioreactor. In this article, a structured model for bioreactions expressed in terms of biological extensive variables is proposed. A biological variable is introduced to calculate the growth rate of the population. The value is updated on the basis of the difference between the composition in the liquid and biotic phase. The structured model is able to predict the transient behavior of different continuous cultures subject to various drastic perturbations. This performance is obtained with a minimum increase in the standard unstructured model complexity (one additional time constant). In the final part, the consequences of decoupling the growth rate from the substrate uptake rate are discussed. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Model identification in presence of incomplete information by generalized principal component analysis: Application to the common and differential responses of Escherichia coli to multiple pulse perturbations in continuous, high-biomass density cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Daniel V. Guebel Abstract In a previous report we described a multivariate approach to discriminate between the different response mechanisms operating in Escherichia coli when a steady, continuous culture of these bacteria was perturbed by a glycerol pulse (Guebel et al., 2009, Biotechnol Bioeng 102: 910,922). Herein, we present a procedure to extend this analysis when multiple, spaced pulse perturbations (glycerol, fumarate, acetate, crotonobetaine, hypersaline plus high-glycerol basal medium and crotonobetaine plus hypersaline basal medium) are being assessed. The proposed method allows us to identify not only the common responses among different perturbation conditions, but to recognize the specific response for a given stimulus even when the dynamics of the perturbation is unknown. Components common to all conditions are determined first by Generalized Principal Components Analysis (GPCA) upon a set of covariance matrices. A metrics is then built to quantify the similitude distance. This is based on the degree of variance extraction achieved for each variable along the GPCA deflation processes by the common factors. This permits a cluster analysis, which recognizes several compact sub-sets containing only the most closely related responsive groups. The GPCA is then run again but is restricted to the groups in each sub-set. Finally, after the data have been exhaustively deflated by the common sub-set factors, the resulting residual matrices are used to determine the specific response factors by classical principal component analysis (PCA). The proposed method was validated by comparing its predictions with those obtained when the dynamics of the perturbation was determined. In addition, it showed to have a better performance than the obtained with other multivariate alternatives (e.g., orthogonal contrasts based on direct GPCA, Tucker-3 model, PARAFAC, etc.). Biotechnol. Bioeng. 2009; 104: 785,795 © 2009 Wiley Periodicals, Inc. [source] The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technologyBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2009Malcolm L. Kennard Abstract The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for the targeted transfection of single or multiple genes and eliminates the need for random integration into native host chromosomes. To illustrate the utility of the ACE System in generating stable, high expressing cell lines, CHO based candidate cell lines were generated to express a human monoclonal IgG1 antibody. Candidate cell lines were generated in under 6 months and expressed over 1,g/L and with specific productivities of up to 45,pg/cell/day under non-fed, non-optimized shake flask conditions. These candidate cell lines were shown to have stable expression of the monoclonal antibody for up to 70 days of continuous culture. The results of this study demonstrate that clonal, stable monoclonal antibody expressing CHO based cell lines can be generated by the ACE System rapidly and perform competitively with those cell lines generated by existing technologies. The ACE System, therefore, provides an attractive and practical alternative to conventional methods of cell line generation. Biotechnol. Bioeng. 2009; 104: 540,553 © 2009 Wiley Periodicals, Inc. [source] Determination of diauxic lag in continuous cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008Dong-Uk Lee Abstract A procedure was developed to characterize diauxic lag of bacteria switching between electron acceptors in continuous culture. In this procedure, a virtual batch growth curve is developed by integrating the time-dependent net specific growth rates of bacteria observed under continuous flow conditions. The length of diauxic lag and the highest net specific growth rate following lag are conveniently estimated from the virtual batch curve. The procedure was found to give reproducible diauxic lag lengths and highest net specific growth rates when applied to experimental data from replicate continuous culture trials. Biotechnol. Bioeng. 2008;101: 642,646. © 2008 Wiley Periodicals, Inc. [source] Stability of recombinant plasmids on the continuous culture of Bifidobacterium animalis ATCC 27536BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Antonio González Vara Abstract Bifidobacterium animalis ATCC 27536 represents among bifidobacteria a host-model for cloning experiments. The segregational and structural stabilities of a family of cloning vectors with different molecular weights but sharing a common core were studied in continuous fermentation of the hosting B. animalis without selective pressure. The rate of plasmid loss (R) and the specific growth rate difference (,,) between plasmid-free and plasmid-carrying cells were calculated for each plasmid and their relationship with plasmid size was studied. It was observed that both R and the numerical value of ,, increased exponentially with plasmid size. The exponential functions correlating the specific growth rate difference and the rate of plasmid loss with the plasmid molecular weight were determined. Furthermore, the smallest of the plasmids studied, pLAV (4.3-kb) was thoroughly characterized by means of its complete nucleotide sequence. It was found that it contained an extra DNA fragment, the first bifidobacterial insertion sequence characterised, named IS 1999. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 145,150, 2003. [source] The effect of continuous culture on the growth and structure of tissue-engineered cartilageBIOTECHNOLOGY PROGRESS, Issue 2 2009Aasma A. Khan Abstract The use of bioreactors for cartilage tissue engineering has become increasingly important as traditional batch-fed culture is not optimal for in vitro tissue growth. Most tissue engineering bioreactors rely on convection as the primary means to provide mass transfer; however, convective transport can also impart potentially unwanted and/or uncontrollable mechanical stimuli to the cells resident in the construct. The reliance on diffusive transport may not necessarily be ineffectual as previous studies have observed improved cartilaginous tissue growth when the constructs were cultured in elevated volumes of media. In this study, to approximate an infinite reservoir of media, we investigated the effect of continuous culture on cartilaginous tissue growth in vitro. Isolated bovine articular chondrocytes were seeded in high density, 3D culture on MillicellÔ filters. After two weeks of preculture, the constructs were cultivated with or without continuous media flow (5,10 ,L/min) for a period of one week. Tissue engineered cartilage constructs grown under continuous media flow significantly accumulated more collagen and proteoglycans (increased by 50,70%). These changes were similar in magnitude to the reported effect of through-thickness perfusion without the need for large volumetric flow rates (5,10,L/min as opposed to 240,800 ,L/min). Additionally, tissues grown in the reactor displayed some evidence of the stratified morphology of native cartilage as well as containing stores of intracellular glycogen. Future studies will investigate the effect of long-term continuous culture in terms of extracellular matrix accumulation and subsequent changes in mechanical function. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Effects of Three-Dimensional Culturing on Osteosarcoma Cells Grown in a Fibrous Matrix: Analyses of Cell Morphology, Cell Cycle, and ApoptosisBIOTECHNOLOGY PROGRESS, Issue 5 2003Chunnuan Chen Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three-dimensional (3-D) fibrous culture had better cell growth and higher metabolic rates than the two-dimensional (2-D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long-term (1 month) T-flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at ,15%. These results could explain why the fibrous bed bioreactor had good long-term stability and was advantageous for production of non-growth-associated proteins by animal cell cultures. [source] Continuous Cultivation of the Diatom Nitzschia laevis for Eicosapentaenoic Acid Production: Physiological Study and Process OptimizationBIOTECHNOLOGY PROGRESS, Issue 1 2002Zhi-You Wen The continuous cultures of the diatom Nitzschia laevis were performed at different dilution rates ( D) and feed glucose concentrations ( S0) to investigate cellular physiological responses and its production potential of eicosapentaenoic acid (EPA). Steady-state cell dry weight, residual glucose concentration, cell growth yield, specific glucose consumption rate, and fatty acid profiles were investigated within the range of D from 0.1 to 1.0 day,1 ( S0 fixed at 20 g/L) and the range of S0 from 5 to 35 g/L ( D fixed at 0.3 day,1), respectively. The highest EPA productivity of 73 mg L -1 day -1 was obtained at D = 0.5 day,1 and S0 = 20 g/L. However, when the continuous culture achieved high productivities of EPA at certain dilution rates and feed glucose concentrations, glucose in the feed could not be consumed completely. Accordingly, the continuous culture was evaluated in terms of both EPA productivity ( P) and glucose assimilation efficiency ( E). The parameter ,, defined as the product of P and E, was used as an overall performance index. Since , is a function of the two independent variables Dand S0, we employed a central composite design to optimize D and S0 for the highest , value. Based on the experimental results of the design, a second-order polynomial equation was established to represent the relationship between , and D and S0. The optimal values of D and S0 were subsequently determined as 0.481 day,1 and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, , value reached 46.5 mg L -1 day -1, suggesting a considerably high efficiency of the continuous culture of N. laevis in terms of EPA production and glucose utilization. [source] Effect of Inoculum Composition and Low KCl Supplementation on the Biological and Rheological Stability of an Immobilized-Cell System for Mixed Mesophilic Lactic Starter ProductionBIOTECHNOLOGY PROGRESS, Issue 6 2001L. Lamboley Two strains of Lactococcus lactissubsp. lactis (L. lactis KB and KBP) and one of L. lactissubsp. lactis biovar. diacetylactis (L. diacetylactis MD) were immobilized separately in ,-carrageenan-locust bean gum gel beads. Continuous fermentations were carried out in supplemented whey permeate in a 1-L pH-controlled stirred tank reactor inoculated with a 30% (v/v) bead inoculum and a bead ratio of 55:30:15 for KB, KBP, and MD, respectively. The process demonstrated a high productivity and microbial stability during the 7-week continuous culture. Compared with previous experiments carried out with an inoculum bead ratio of 33:33:33 for KB, KBP, and MD beads, respectively, the modification of the inoculum bead ratio had apparently little effect on free and immobilized, total and specific populations. A dominant behavior of L. diacetylactis MD over the other strains of the mixed culture was observed both with free-cell populations in the effluent and with immobilized-cell populations. Additional experiments were carried out with other strain combinations for continuous inoculation-prefermentation of milk. The data also confirmed the dominance of L. diacetylactis during long-term continuous immobilized-cell fermentations. This dominance may be tentatively explained by the local competition involved in the development of the bead cross-contamination and in citrate utilization by L. diacetylactis strains. The gel beads demonstrated a high rheological stability during the 7-week continuous fermentation even at low KCl supplementation of the broth medium (25 mM KCl). [source] Transforming Growth Factor-, Induces the Differentiation of Sarcomatoid Cholangiocarcinoma CellsCANCER SCIENCE, Issue 2 2000Munechika Enjoji A sarcomatoid cholangiocarcinoma cell line, ETK-1, was established from a patient. Phenotypically, the cells corresponded to immature biliary epithelial cells. Because a small number of ETK-1 cells appeared to differentiate spontaneously along a biliary epithelial lineage in continuous culture, we examined the factors that initiate and/or promote the differentiation of the cells. Transforming growth factor-, (TGF,) induced significant changes in ETK-1 cells. After stimulation with the factor, ETK-1 cells displayed morphologic transformation at a much higher frequency, with the appearance of many large cells with intracytoplasmic vacuoles, and the production of mucinous substances. These morphologically transformed cells were phenotypically similar to welldifferentiated adenocarcinoma cells. The expression pattern of integrins after TGF, treatment also supported the maturation of the ETK-1 cells. The antibody against the receptor of TGF, inhibited these changes by TGF,. Moreover, the proliferation rate of ETK-1 cells was suppressed by TGF,. Our data suggest that TGF, can act as a differentiation factor along a biliary epithelial lineage. [source] Role of reserve carbohydrates in the growth dynamics of Saccharomyces cerevisiae,FEMS YEAST RESEARCH, Issue 8 2004Vincent Guillou Abstract The purpose of this study was to explore the role of glycogen and trehalose in the ability of Saccharomyces cerevisiae to respond to a sudden rise of the carbon flux. To this end, aerobic glucose-limited continuous cultures were challenged with a sudden increase of the dilution rate from 0.05 to 0.15 h,1. Under this condition, a rapid mobilization of glycogen and trehalose was observed which coincided with a transient burst of budding and a decrease of cell biomass. Experiments carried out with mutants defective in storage carbohydrates indicated a predominant role of glycogen in the adaptation to this perturbation. However, the real importance of trehalose in this response was veiled by the unexpected phenotypes harboured by the tps1 mutant, chosen for its inability to synthesize trehalose. First, the biomass yield of this mutant was 25% lower than that of the isogenic wild-type strain at dilution rate of 0.05 h,1, and this difference was annulled when cultures were run at a higher dilution rate of 0.15 h,1. Second, the tps1 mutant was more effective to sustain the dilution rate shift-up, apparently because it had a faster glycolytic rate and an apparent higher capacity to consume glucose with oxidative phosphorylation than the wild type. Consequently, a tps1gsy1gsy2 mutant was able to adapt to the dilution rate shift-up after a long delay, likely because the detrimental effects from the absence of glycogen was compensated for by the tps1 mutation. Third, a glg1,glg2, strain, defective in glycogen synthesis because of the lack of the glycogen initiation protein, recovered glycogen accumulation upon further deletion of TPS1. This recovery, however, required glycogen synthase. Finally, we demonstrated that the rapid breakdown of reserve carbohydrates triggered by the shift-up is merely due to changes in the concentrations of hexose-6-phosphate and UDPglucose, which are the main metabolic effectors of the rate-limiting enzymes of glycogen and trehalose pathways. [source] Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous culturesFEMS YEAST RESEARCH, Issue 2 2003Helena Albergaria Abstract The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures. By both cultivation techniques this yeast was found to be Crabtree-positive. Up to a dilution rate of 0.25 h,1, glucose was completely metabolised into biomass, glycerol and carbon dioxide. Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids. Biomass yield during the purely oxidative growth was 0.49 g g,1 and decreased to 0.26 g g,1 for D=0.34 h,1. A maximal specific ethanol production rate of 1.36 mmol g,1 h,1 and a maximal ethanol yield of 0.05 g g,1 were achieved at D=0.34 h,1. [source] Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2002Jasper A. Diderich Abstract In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate. [source] Fructose and glucose mediates enterotoxin production and anaerobic metabolism of Bacillus cereus ATCC14579TJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2009O. Ouhib-Jacobs Abstract Aims:, To determine the effects of carbohydrates on Bacillus cereus ATCC14579T anaerobic metabolism and enterotoxin production in amino acids rich medium. Methods and Results:,Bacillus cereus anaerobic growth on different carbohydrates (glucose, fructose, sucrose or glucose,fructose mixture) was examined in synthetic mMOD medium under continuous cultures (, = 0·2 h,1). Fermentation end-products, flux partitioning at each key branch points of the mixed acid pathway and consumption or production of amino acids were determined. On both fructose and sucrose, ATP production was favoured via acetate production from acetyl-CoA. In addition, amino acids present in the growth medium showed significant variations with high consumption of serine and net production of glutamate and alanine on some or all sugars. Enterotoxins Hbl and Nhe production was high during growth on fructose (or mixtures involving a fructose moiety). Conclusions:, Fructose was identified as a key sugar influencing anaerobic metabolism and toxin production of B. cereus. Significance and Impact of the Study:, The physiological differences associated with the fermentation of the various carbohydrates clearly modify toxinogenesis indicating that the risk of foodborne pathogens is to some extent dependent upon the prevailing nutritional environment. [source] A structured model for the simulation of bioreactors under transient conditionsAICHE JOURNAL, Issue 11 2009Jérôme Morchain Abstract Modeling the transient behavior of continuous culture is of primary importance for the scale-up of biological processes. Spatial heterogeneities increase with the reactor size and micro-organisms have to cope with a fluctuating environment along their trajectories within the bioreactor. In this article, a structured model for bioreactions expressed in terms of biological extensive variables is proposed. A biological variable is introduced to calculate the growth rate of the population. The value is updated on the basis of the difference between the composition in the liquid and biotic phase. The structured model is able to predict the transient behavior of different continuous cultures subject to various drastic perturbations. This performance is obtained with a minimum increase in the standard unstructured model complexity (one additional time constant). In the final part, the consequences of decoupling the growth rate from the substrate uptake rate are discussed. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] NITROGEN LIMITATION EFFECTS OF DIFFERENT NITROGEN SOURCES ON NUTRITIONAL QUALITY OF TWO FRESHWATER ORGANISMS, SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AND SYNECHOCOCCUS SP. (CYANOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 5 2003Gunnel Ahlgren Food quality for grazers has been related to mineral (nitrogen, phosphorus) and biochemical (amino acids, fatty acids) constituents. The aim of the study was to examine the influence of different nitrogen sources on these constituents in two organisms, the green alga Scenedesmus quadricauda Turp. and the cyanobacterium Synechococcus sp., commonly used in feeding experiments. The two organisms were grown in continuous cultures at different growth rates. Nitrate or ammonium salts were used as nitrogen sources under both replete and limited conditions. Carbon content (mg·g,1 dry weight) was stable in both organisms independent of nitrogen source, nitrogen limitation, and growth rate. Nitrogen content decreased with limitation and growth rate in Scenedesmus and to a lesser degree in Synechococcus, whereas changes in phosphorus content were not statistically significant. The relative proportions of amino acids (% of total amino acids) were relatively stable in both organisms, whereas the proportions of fatty acids varied with growth rate and limitation. Fatty acid content was much lower in Synechococcus than in Scenedesmus. At N limitation, polyunsaturated fatty acids (PUFAs) showed lower levels in both organisms. The change occurred in the ,3 PUFA (linolenic acid) of the green alga and in the ,6 PUFA (linoleic acid) of the cyanobacterium. The difference in the response of N limitation in the two organisms may be traced to the different composition of the chloroplast membranes (the prokaryotic way) and the microsomal membranes (the eukaryotic way) where the desaturation takes place. [source] | ||||||||||||||||||||||||||||