Showing papers on "Chemostat published in 2015"

A Single Dynamic Metabolic Model Can Describe mAb Producing CHO Cell Batch and Fed-Batch Cultures on Different Culture Media

Abstract: CHO cell culture high productivity relies on optimized culture medium management under fed-batch or perfused chemostat strategies enabling high cell densities. In this work, a dynamic metabolic model for CHO cells was further developed, calibrated and challenged using datasets obtained under four different culture conditions, including two batch and two fed-batch cultures comparing two different culture media. The recombinant CHO-DXB11 cell line producing the EG2-hFc monoclonal antibody was studied. Quantification of extracellular substrates and metabolites concentration, viable cell density, monoclonal antibody concentration and intracellular concentration of metabolite intermediates of glycolysis, pentose-phosphate and TCA cycle, as well as of energetic nucleotides, were obtained for model calibration. Results suggest that a single model structure with a single set of kinetic parameter values is efficient at simulating viable cell behavior in all cases under study, estimating the time course of measured and non-measured intracellular and extracellular metabolites. Model simulations also allowed performing dynamic metabolic flux analysis, showing that the culture media and the fed-batch strategies tested had little impact on flux distribution. This work thus paves the way to an in silico platform allowing to assess the performance of different culture media and fed-batch strategies.

Physiological, biomass elemental composition and proteomic analyses of Escherichia coli ammonium-limited chemostat growth, and comparison with iron- and glucose-limited chemostat growth

Abstract: Escherichia coli physiological, biomass elemental composition and proteome acclimations to ammonium-limited chemostat growth were measured at four levels of nutrient scarcity controlled via chemostat dilution rate. These data were compared with published iron- and glucose-limited growth data collected from the same strain and at the same dilution rates to quantify general and nutrient-specific responses. Severe nutrient scarcity resulted in an overflow metabolism with differing organic byproduct profiles based on limiting nutrient and dilution rate. Ammonium-limited cultures secreted up to 35 % of the metabolized glucose carbon as organic byproducts with acetate representing the largest fraction; in comparison, iron-limited cultures secreted up to 70 % of the metabolized glucose carbon as lactate, and glucose-limited cultures secreted up to 4 % of the metabolized glucose carbon as formate. Biomass elemental composition differed with nutrient limitation; biomass from ammonium-limited cultures had a lower nitrogen content than biomass from either iron- or glucose-limited cultures. Proteomic analysis of central metabolism enzymes revealed that ammonium- and iron-limited cultures had a lower abundance of key tricarboxylic acid (TCA) cycle enzymes and higher abundance of key glycolysis enzymes compared with glucose-limited cultures. The overall results are largely consistent with cellular economics concepts, including metabolic tradeoff theory where the limiting nutrient is invested into essential pathways such as glycolysis instead of higher ATP-yielding, but non-essential, pathways such as the TCA cycle. The data provide a detailed insight into ecologically competitive metabolic strategies selected by evolution, templates for controlling metabolism for bioprocesses and a comprehensive dataset for validating in silico representations of metabolism.

The Influence of Growth Rate on 2H/1H Fractionation in Continuous Cultures of the Coccolithophorid Emiliania huxleyi and the Diatom Thalassiosira pseudonana

Abstract: The hydrogen isotope (2H/1H) ratio of lipids from phytoplankton is a powerful new tool for reconstructing hydroclimate variations in the geologic past from marine and lacustrine sediments. Water 2H/1H changes are reflected in lipid 2H/1H changes with R2 > 0.99, and salinity variations have been shown to cause about a 1‰ change in lipid δ2H values per unit (ppt) change in salinity. Less understood are the effects of growth rate, nutrient limitation and light on 2H/1H fractionation in phytoplankton. Here we present the first published study of growth rate effects on 2H/1H fractionation in the lipids of coccolithophorids grown in continuous cultures. Emiliania huxleyi was cultivated in steady state at four growth rates and the δ2H value of individual alkenones (C37:2, C37:3, C38:2, C38:3), fatty acids (C14:0, C16:0, C18:0), and 24-methyl cholest-5,22-dien-3β-ol (brassicasterol) were measured. 2H/1H fractionation increased in all lipids as growth rate increased by 24‰ to 79‰ (div d-1)-1. We attribute this response to a proportional increase in the fraction of NADPH from Photosystem I (PS1) of photosynthesis relative to NADPH from the cytosolic oxidative pentose phosphate (OPP) pathway in the synthesis of lipids as growth rate increases. A 3-endmember model is presented in which lipid hydrogen comes from NADPH produced in PS1, NADPH produced by OPP, and intracellular water. With published values or best estimates of the fractionation factors for these sources (αPS1 = 0.4, αOPP = 0.75, and αH2O = 0) and half of the hydrogen in a lipid derived from water the model indicates αlipid = 0.79. This value is within the range measured for alkenones (αalkenone = 0.77 to 0.81) and fatty acids (αFA = 0.75 to 0.82) in the chemostat cultures, but is greater than the range for brassicasterol (αbrassicasterol = 0.68 to 0.72). The latter is attributed to a greater proportion of hydrogen from NADPH relative to water in isoprenoid lipids. The model successfully explains the increase in 2H/1H fractionation in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol from marine centric diatom T. pseudonana chemostat cultures as growth rate increases. Insensitivity of αFA in those same cultures may be attributable to a larger fraction of hydrogen in fatty acids sourced from intracellular water at the expense of NADPH as growth rate increases. The high sensitivity of α to growth rate in E. huxleyi lipids and a T. pseudonana sterol implies that any change in growth rate larger than ~0.15 div d-1 can cause a change in δ2Hlipid that is larger than the analytical error of the measurement (~5‰), and needs to be considered when interpreting δ2Hlipid variations in sediments.

Growth-rate dependency of de novo resveratrol production in chemostat cultures of an engineered Saccharomyces cerevisiae strain

Abstract: Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures. Stoichiometric analysis revealed that de novo resveratrol production from glucose requires 13 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g. 27 % at 0.03 h−1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in chemostat. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g. ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer. De novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.

Physiological and cell morphology adaptation of Bacillus subtilis at near‐zero specific growth rates: a transcriptome analysis

Abstract: Nutrient scarcity is a common condition in nature, but the resulting extremely low growth rates (below 0.025 h(-1) ) are an unexplored research area in Bacillus subtilis. To understand microbial life in natural environments, studying the adaptation of B. subtilis to near-zero growth conditions is relevant. To this end, a chemostat modified for culturing an asporogenous B. subtilis sigF mutant strain at extremely low growth rates (also named a retentostat) was set up, and biomass accumulation, culture viability, metabolite production and cell morphology were analysed. During retentostat culturing, the specific growth rate decreased to a minimum of 0.00006 h(-1) , corresponding to a doubling time of 470 days. The energy distribution between growth and maintenance-related processes showed that a state of near-zero growth was reached. Remarkably, a filamentous cell morphology emerged, suggesting that cell separation is impaired under near-zero growth conditions. To evaluate the corresponding molecular adaptations to extremely low specific growth, transcriptome changes were analysed. These revealed that cellular responses to near-zero growth conditions share several similarities with those of cells during the stationary phase of batch growth. However, fundamental differences between these two non-growing states are apparent by their high viability and absence of stationary phase mutagenesis under near-zero growth conditions.

Defining the nitrogen regulated transcriptome of Mycobacterium smegmatis using continuous culture

Abstract: Nitrogen is essential for microbial growth and its importance is demonstrated by the complex regulatory systems used to control the transport, assimilation and metabolism of nitrogen. Recent studies are beginning to shed light on how mycobacteria respond to nitrogen limitation and several regulators (e.g., GlnR, PII) have been characterized at a molecular level. However, despite this progress, our knowledge of the transcriptional response of mycobacteria to nitrogen limitation and its regulation is confined to batch culture. To gain further insight into the response of mycobacteria to nitrogen limitation, we developed a nitrogen-limited chemostat. We compared the transcriptional response of nitrogen-limited cells to carbon-limited cells using RNA-seq analysis in a continuous culture model at a constant growth rate. Our findings revealed significant changes in the expression of 357 genes (208 upregulated, 149 downregulated; >2-fold change, false discovery rate <5 %) in response to nitrogen limitation in continuous culture. The vast majority of the GlnR regulon (68 %) was differentially expressed under nitrogen limitation in continuous culture and approximately 52 % of the 357 genes overlapped with a previously published study investigating the response of M. smegmatis to nitrogen limitation in batch culture, while expression of only 17 % of the genes identified in batch culture were affected in our chemostat model. Moreover, we identified a unique set of 45 genes involved in the uptake and metabolism of nitrogen that were exclusive to our chemostat model. We observed strong downregulation of pathways for amino acid catabolism (i.e., alanine, aspartate, valine, proline and lysine), suggesting preservation of these amino acids for critical cellular function. We found 16 novel transcriptional regulators that were directly or indirectly involved in the global transcriptomic response of M. smegmatis to nitrogen limitation and identified several non-coding RNAs that might be involved in the transcriptional or post-transcriptional regulation of nitrogen-regulated gene expression. Using nitrogen-limited continuous culture we identified the nitrogen-responsive transcriptome of M. smegmatis, including a number of small non-coding RNAs implicated in controlling nitrogen-regulated gene expression.

Applying systems biology tools to study n‐butanol degradation in Pseudomonas putida KT2440

Abstract: To smoothen the process of n-butanol formation in Pseudomonas putida KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance. Here, we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. Pseudomonas putida KT2440 was cultivated in multiple chemostat fermentations using n-butanol either as sole carbon source or together with glucose. Pseudomonas putida KT2440 revealed maximum growth rates (μ) of 0.3 h−1 with n-butanol as sole carbon source and of 0.4 h−1 using equal C-molar amounts of glucose and n-butanol. While C-mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when n-butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when n-butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing n-butanol consumption. 13C flux analysis revealed that central metabolism was split into a glucose-fueled Entner–Doudoroff/pentose-phosphate pathway and an n-butanol-fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of n-butanol could be unraveled, thus representing an important basis for rendering P. putida KT2440 from an n-butanol consumer to a producer in future metabolic engineering studies.

Growth retardation of Escherichia coli by artificial increase of intracellular ATP

Abstract: Overexpression of phosphoenolpyruvate carboxykinase (PCK) was reported to cause the harboring of higher intracellular ATP concentration in Escherichia coli, accompanied with a slower growth rate. For systematic determination of the relationship between the artificial increase of ATP and growth retardation, PCKWT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60 % greater catalytic efficiency than that of PCKWT. Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 μmol/gcell, with the slowest growth rate of 0.66 h−1, while the PCKWT-overexpressing cells displayed 1.00 μmol/gcell ATP concentration with the growth rate of 0.84 h−1 and the control had 0.28 μmol/gcell with 1.03 h−1. To find a plausible reason, PCK-overexpressing cells in a steady state during chemostat growth were applied to monitor intracellular reactive oxygen species (ROS). Higher amount of intracellular ROS were observed as the ATP levels increased. To confirm the hypothesis of slower growth rate without perturbation of the carbon flux by PCK-overexpression, phototrophic Gloeobacter rhodopsin (GR) was expressed. The GR-expressing strain under illumination harbored 81 % more ATP concentration along with 82 % higher ROS, with a 54 % slower maximum growth rate than the control, while both the GR-expressing strain under dark and dicarboxylate transporter (a control membrane protein)-expressing strain showed a lower ATP and increased ROS, and slower growth rate. Regardless of carbon flux changes, the artificial ATP increase was related to the ROS increase and it was reciprocally correlated to the maximum growth rate. To verify that the accumulated intracellular ROS were responsible for the growth retardation, glutathione was added to the medium to reduce the ROS. As a result, the growth retardation was restored by the addition of 0.1 mM glutathione. Anaerobic culture even enabled the artificial ATP-increased E. coli to grow faster than control. Collectively, it was concluded that artificial ATP increases inhibit the growth of E. coli due to the overproduction of ROS.

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

Abstract: The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.

Chemostats with random inputs and wall growth

Abstract: Chemostat refers to a laboratory device used for growing microorganisms in a cultured environment and has been regarded as an idealization of nature to study competition modeling in mathematical biology. The simple form of chemostat model assumes that the availability of nutrient and its supply rate are both fixed. In addition, the tendency of microorganisms to adhere to surfaces is neglected by assuming the flow rate is fast enough. However, these assumptions largely limit the applicability of chemostat models to realistic competition systems. In this paper, we relax these assumptions and study chemostat models with random nutrient supplying rate or random input nutrient concentration, with or without wall growth. This leads to random dynamical systems and requires the concept of random attractors developed in the theory of random dynamical systems. Our results include existence of uniformly bounded non-negative solutions, existence of random attractors, and geometric details of random attractors for different value of parameters. Copyright © 2015 John Wiley & Sons, Ltd.

Near-zero growth kinetics of Pseudomonas putida deduced from proteomic analysis

Abstract: Intensive microbial growth typically observed in laboratory rarely occurs in nature. Because of severe nutrient deficiency, natural populations exhibit near-zero growth (NZG). There is a long-standing controversy about sustained NZG, specifically whether there is a minimum growth rate below which cells die or whether cells enter a non-growing maintenance state. Using chemostat with cell retention (CCR) of Pseudomonas putida, we resolve this controversy and show that under NZG conditions, bacteria differentiate into growing and VBNC (viable but not non-culturable) forms, the latter preserving measurable catabolic activity. The proliferating cells attained a steady state, their slow growth balanced by VBNC production. Proteomic analysis revealed upregulated (transporters, stress response, self-degrading enzymes and extracellular polymers) and downregulated (ribosomal, chemotactic and primary biosynthetic enzymes) proteins in the CCR versus batch culture. Based on these profiles, we identified intracellular processes associated with NZG and generated a mathematical model that simulated the observations. We conclude that NZG requires controlled partial self-digestion and deep reconfiguration of the metabolic machinery that results in the biosynthesis of new products and development of broad stress resistance. CCR allows efficient on-line control of NZG including VBNC production. A well-nuanced understanding of NZG is important to understand microbial processes in situ and for optimal design of environmental technologies.

Effect of dilution rate and methanol-glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut(+) phenotype in continuous culture

Abstract: The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol-glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol-glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04-0.06 h(-1) , while ROL yield decreased along the whole dilution rate range evaluated (0.03-0.1 h(-1) ). Compared to production level achieved with methanol-only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g-biomass(-1) h(-1) ), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut(+) phenotype for heterologous protein production.

An array microhabitat system for high throughput studies of microalgal growth under controlled nutrient gradients

Abstract: Microalgae have been increasingly recognized in the fields of environmental and biomedical engineering because of its use as base materials for biofuels or biomedical products, and also the urgent needs to control harmful algal blooms protecting water resources worldwide. Central to the theme is the growth rate of microalgae under the influences of various environmental cues including nutrients, pH, oxygen tension and light intensity. Current microalgal culture systems, e.g. raceway ponds or chemostats, are not designed for system parameter optimizations of cell growth. In this article, we present the development of an array microfluidic system for high throughput studies of microalgal growth under well defined environmental conditions. The microfluidic platform consists of an array of microhabitats flanked by two parallel side channels, all of which are patterned in a thin agarose gel membrane. The unique feature of the device is that each microhabitat is physically confined suitable for both motile and non-motile cell culture, and at the same time, the device is transparent and can be perfused through the two side channels amendable for precise environmental control of photosynthetic microorganisms. This microfluidic system is used to study the growth kinetics of a model microalgal strain, Chlamydomonas reinhardtii (C. reinhardtii), under ammonium (NH4Cl) concentration gradients. Experimental results show that C. reinhardtii follows Monod growth kinetics with a half-saturation constant of 1.2 ± 0.3 μM. This microfluidic platform provides a fast (~50 fold speed increase), cost effective (less reagents and human intervention) and quantitative technique for microalgal growth studies, in contrast to the current chemostat or batch cell culture system. It can be easily extended to investigate growth kinetics of other microorganisms under either single or co-culture setting.

Global dynamics of the buffered chemostat for a general class of response functions.

Abstract: We study how a particular spatial structure with a buffer impacts the number of equilibria and their stability in the chemostat model We show that the occurrence of a buffer can allow a species to persist or on the opposite to go extinct, depending on the characteristics of the buffer For non-monotonic response functions, we characterize the buffered configurations that make the chemostat dynamics globally asymptotically stable, while this is not possible with single, serial or parallel vessels of the same total volume and input flow These results are illustrated with the Haldane kinetic function

Demonstration of a two-stage aerobic/anaerobic chemostat for the enhanced production of hydrogen and biomass from unicellular nitrogen-fixing cyanobacterium

Abstract: Due to incompatible demands between the cyanobacterial growth and its sequential hydrogen (H 2 ) photoproduction mechanism, a two-stage chemostat photobioreactor (PBR) system was employed to cultivate a unicellular, nitrogen-fixing cyanobacterium Cyanothece sp. ATCC 51142. Our developed system, consisting of a series of two physically separated PBRs, has been operated non-stop for consecutive 750 h (~ 31 days), without any losses in its performance. Based on nutrient kinetics determined in batch PBR, a dilution rate of 0.015 h − 1 was chosen to replenish the culture with all of essential substrates, while not interrupting the H 2 formation reaction. The physiological steady-state condition was achieved on day 12. The dry biomass concentrations, accumulated in the primary growth and the secondary H 2 -production PBRs, reached the final values of 2 and 1.5 g L −1 respectively. By comparing to a single-stage batch system, the chemostat displayed more than 6.4 times higher H 2 productivity and 7.3 folds greater biomass yield. The steady-state conversion from glycerol into H 2 was ~ 28.3%. The stationary growth phase was also observed when the nutrient-replete culture eventually became under light-limited condition. Additionally, glycerol was reproducibly demonstrated as an effective anaerobic-inducer onto an air-incubated culture of this Cyanothece strain.

Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures

Abstract: Background Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum. Methods In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD) processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA) were carried out at two temperatures (37°C and 33°C) and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system) or ERAD II (Autophagosoma/Lisosomal system) pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1) and low (0.012 h-1) dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA). Results and Conclusion Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO cells is modulated predominantly by specific growth rate, indicating that the culture temperature has a lower weighted effect within the range of conditions evaluated in this work.

Chemostats with time-dependent inputs and wall growth

Abstract: Traditional assumptions in the simple chemostat model include fixed availability of the nutrient and its supply rate, an d fast flow rate to avoid wall growth. However, these assumptio ns become unrealistic when the availability of a nutrient depends on the nutrient consumption rate and input nutrient concentration and when the flow rate is not fast enough. In this paper, we rel ax these assumptions and study the chemostat models with a variable nutrient supplying rate or a variable input nutrient concent ration, with or without wall growth. This leads the models to nonautonomous dynamical systems and requires new concepts of nonautonomous attractors from the recently developed theory of nonautonomous dynamical systems. Our results provide sufficient cond itions for existence of nonautonomous attractors and singleton attractors.

Method for treating solid wastes by culturing biological leachate by use of membrane bioreactor

Abstract: The invention relates to a method for treating solid wastes by culturing biological leachate by use of a membrane bioreactor, and belongs to the field of a solid waste resource treatment technology The membrane bioreactor is applied to biological leachate culture and regeneration firstly and is used for the innocent and resource treatment of dangerous solid wastes A group of membrane components is arranged in the bioreactor, and the metabolism and the growth of leaching bacterial strains are controlled through the adjustment of the aeration rate, the stirring speed, the concentration of nutrient substances and the like, when the concentration of the leaching bacterial strains is in a stable phase, water inlet and water outlet functions are started to enable the membrane bioreactor to be in a chemostat state, the leaching bacterial strains can be enriched by the interception action of the membrane, and the yield of the biological leachate is increased The method can be used for effectively solving the problems that the leaching bacterial strains grow slowly, the biomass is low, the leaching efficiency is low, the leaching period is long and the like, has the beneficial effects of simpleness, high safety, energy conservation and wide application range, can be operated conveniently and is suitable for the innocent and resource treatment of different solid wastes

Chemostat production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934.

Abstract: Production of the bacteriocin pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in pH-controlled batch and chemostat cultures using a complex medium containing glucose, sucrose or fructose. In chemostat cultures operated at 150 rpm, 30°C, 60% dissolved oxygen tension, pH 6.5, and D = 0.148 h(-1) , the pediocin titer reached 185 AU/mL representing an increase of 32% compared with batch cultures in which glucose was used as the carbon source. Pediocin biosynthesis was markedly affected by the growth rate of the producer microorganism. For all carbon sources tested, pediocin production appeared to take place only at dilution rates lower than μmax . However, only glucose supported production at the very low dilution rate of 0.05 h(-1) indicating a direct regulation of pediocin biosynthesis by the carbon source. Glucose supported higher biomass productivity and higher pediocin titers and yields compared with the other sugars used.

A biological treatment of industrial wastewaters: contois kinetics

Abstract: This paper analyses the steady-state operation of a generalized bioreactor model that encompasses a continuous-flow bioreactor and an idealized continuous-flow membrane bioreactor as limiting cases. A biodegradation of organic materials is modelled using Contois growth kinetics. The bioreactor performance is analysed by finding the steady-state solutions of the model and determining their stability as a function of the dimensionless residence time. We show that an effective recycle parameter improves the performance of the bioreactor at moderate values of the dimensionless residence time. However, at sufficiently large values of the dimensionless residence time, the performance of the bioreactor is independent of the recycle ratio. doi:10.1017/S144618111500005X

Competition between two similar species in the unstirred chemostat

Abstract: This paper deals with the competition between two similar species in the unstirred chemostat. Due to the strict competition of the unstirred chemostat model, the global dynamics of the system is attained by analyzing the equilibria and their stability. It turns out that the dynamics of the system essentially depends upon certain function of the growth rate. Moreover, one of the semi-trivial stationary solutions or the unique coexistence steady state is a global attractor under certain conditions. Biologically, the results indicate that it is possible for the mutant to force the extinction of resident species or to coexist with it.

Modeling perfusion at small scale using ambr15TM

Abstract: Reaching cell densities higher than 80 million with the minimum possible perfusion rates is a goal for an increasing proportion of processes developed by biopharmaceutical companies. With the goal of fulfilling the industry needs for better commercial and customized perfusion media, SAFC evaluated different small-scale perfusion models to achieve an efficient work flow that can accommodate perfusion systems. SAFC successfully uses an optimized work flow for the development of media and feeds for fed-batch cell culture that integrates high-throughput screening, statistical tools and bench-top bioreactor scale studies. In this model, 96-deep well plates are used for the initial high throughput screening, followed by further development in spin tubes or shake flasks. At this time, there is no commercially available cell separation device that can be used for scales of 30mL or lower. The application of the 96-deep well plate or spin tubes model for perfusion showed to have severe limitations, specifically when trying to optimize processes to extremely low cell specific perfusion rates (CSPR). In order to develop media that can sustain the desired high densities and productivity at the desired low CSPRs, we needed a representative model that provided enough throughput to apply our statistical analysis. With this goal, we evaluated an alternative small scale model using the automation and process control offered by the ambr15TM. In this work, we show how ambr15TM fits in the work flow for perfusion media development and its comparability to a chemostat bioreactor. Conclusions and future work • The use of batch TPPs or well plates allows for high throughput screening that can be used for the selection of media to be used for perfusion. However, this work shows that observations made on TPPs do not always transfer to a chemostat system. It is not known at this time if this observation is due to differences between a dynamic and a continuous process or due to process parameters such as pH and DO and this is currently being investigated. • Differences when ranking different media based on their specific growth and productivity between TPPs and ambr15TM were found. The ranking in specific productivity was more similar between TPPs and ambr15TM if the comparison is made on the growth phase of the chemostat. While the absolute values are not comparable (Fig. 3B) the ranking and behavior on the stability of productivity was comparable (Fig. 1C and 2B). This fact underlines the need for a model that can represent a steady state as opposed to a dynamic model. • The chemostat run in ambr15TM is a semi-chemostat. Because of the discontinuity of the model when compared to a fully continuous process, limitations on growth and potentially productivity can become visible at higher dilution rates than in bioreactors (Figure 3A and B). • Work published by Heltmann (2015) and Henry et. al (2008) showed comparability in specific rates between chemostat and perfusion at similar CSPR. In this work we showed that ambr15TM can be used as a tool to model a chemostat and evaluate different media that ultimately is going to be used in perfusion mode. We expect that this model is going to allow us to evaluate media with very different characteristics in order to perform media component optimization at a relatively high throughput. Figure 1. High seed density batch culture • Peak cell densities between 10-15x106vc/mL were observed. Culture longevity was inversely correlated to the peak cell density (Fig. 1A) • Cells stopped producing at the peak density with the exception of M4 (Fig. 1B). • Specific growth rates ranked as M3 > M4> M1 > M2 (data not shown) • Specific productivity ranked as M4 > M2 ≥ M1 > M3 (Fig. 1C) CSPR: cell specific perfusion rate (nL/cell*d) D: dilution rate (vvd) F: volume of medium exchanged (mL) IVCD: integral viable cell density (cell*d/mL) μ: growth rate (d-1) N: number of media exchanges per day P: IgG concentration (mg/L) qp: specific productivity (pcd) X: cell density (vc/mL) V: working volume (mL) Small scale models

Study of two-nutrient and two-micro-organism chemostat model with pulsed input in a polluted environment

Abstract: In this paper, a model of Beddington–DeAngelies chemostat involving two species of micro-organism competing for two perfectly complementary growth-limiting nutrients and pulsed input of toxicant in the polluted environment was studied. Using Floquet theory and small amplitude perturbation method, a conclusion was that there exists two-micro-organism eradication periodic solution and which is global asymptotical stability. At the same time, the condition of the permanence for system was obtained. From the biological point of view, the method for protecting species is to improve the amount of impulsive period, and control the amount of toxicant input to the chemostat. Finally, our results are illustrated by numerical simulations.