RESUMEN
The paper describes the purification, biochemical characterization, sequence determination, and classification of a novel thermophilic hydrolase from Thermobifida fusca (TfH) which is highly active in hydrolyzing aliphatic-aromatic copolyesters. The secretion of the extracellular enzyme is induced by the presence of aliphatic-aromatic copolyesters but also by adding several other esters to the medium. The hydrophobic enzyme could be purified applying a combination of (NH(4))SO(4)-precipitation, cation-exchange chromatography, and hydrophobic interaction chromatography. The 28 kDa enzyme exhibits a temperature maximum of activity between 65 and 70 degrees C and a pH maximum between pH 6 and 7 depending on the ion strength of the solution. According to the amino sequence determination, the enzyme consists of 261 amino acids and was classified as a serine hydrolase showing high sequence similarity to a triacylglycerol lipase from Streptomyces albus G and triacylglycerol-aclyhydrolase from Streptomyces sp. M11. The comparison with other lipases and esterases revealed the TfH exhibits a catalytic behavior between a lipase and an esterase. Such enzymes often are named as cutinases. However, the results obtained here show, that classifying enzymes as cutinases seems to be generally questionable.
Asunto(s)
Actinomycetales/enzimología , Hidrolasas/química , Poliésteres/química , Secuencia de Aminoácidos , Biodegradación Ambiental , Concentración de Iones de Hidrógeno , Hidrolasas/clasificación , Hidrolasas/aislamiento & purificación , Datos de Secuencia Molecular , Temperatura , Factores de TiempoRESUMEN
The reductive biotransformation of mercuric ions to elemental mercury was studied by applying a model system with a genetically engineered Pseudomonas putida strain in a lab scale three-phase fluidized bed (TPFB). The aim was to demonstrate the suitability of the TPFB to demercurize effluent streams containing up to 10 mg Hg2+ dm(-3). The TPFB is used, first, to carry out the biotransformation on the alginate immobilized biocatalyst and, second, to remove the produced Hg0 by volatilization into the gas phase followed by its recovery through fast oxidative absorption. Targeted experiments with the immobilized biocatalyst were designed and carried out to determine mercury adsorption data on the biomass and all relevant mass transport rates at conditions prevailing in the TPFB. The evaluation of the performance data in the TPFB revealed almost complete reaction control and hence negligibility of mass transfer resistances. This simplifies the scale-up of larger TPFB reactors for mercury removal as it can be based on the known kinetics alone. The measured biotransformation capacities in the TPFB are similar to those reported for the fixed bed technology which has already proven its applicability at an industrial scale in long time runs. However, the TPFB offers some advantages over the fixed bed and could therefore possibly be a favorable, reliable, and less costly alternative to the existing technology.
Asunto(s)
Reactores Biológicos , Contaminantes Ambientales/metabolismo , Mercurio/metabolismo , Biomasa , Biotransformación , Control de Costos , Contaminantes Ambientales/aislamiento & purificación , Ingeniería Genética , Mercurio/aislamiento & purificación , Pseudomonas putida/genética , Pseudomonas putida/fisiologíaRESUMEN
Alginate, a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid and currently commercially produced from the marine brown algae, can also be biologically produced by bacteria such as Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas. The ever-increasing applications of this polymer in the food and pharmaceutical sectors have led to continuing research interest aimed at better understanding the metabolic pathways, the physiological or biological function of this polymer, the regulation of its formation and composition, and optimising the microbial production process. These aspects are reviewed here, with particular attention to alginate formation in the soil bacterium A. vinelandii. In addition, the biotechnological and industrial applications of alginate are summarised.
Asunto(s)
Alginatos/química , Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , Biotecnología/métodos , Azotobacter vinelandii/crecimiento & desarrollo , Industria Farmacéutica/métodos , Industria de Alimentos/métodosRESUMEN
The biological degradation behaviour of the aliphatic-aromatic copolyester Ecoflex was investigated with regard to the degree of degradation and the intermediates formed during the degradation process. The individual thermophilic strain Thermomonospora fusca, isolated from compost material, was used for the degradation experiments in a defined synthetic medium at 55 degrees C. After 22 days of degradation more than 99.9% of the polymer had depolymerized and with regard to the degradation of the diacid and diol components of Ecoflex only the monomers of the copolyesters (1,4-butanediol, terephthalate and adipate) could be detected by gas chromatography/mass spectroscopy (GC-MS) measurements in the medium. In interrupted degradation experiments predominantly the monoesters of adipic acid and terephthalic acid with 1,4-butanediol were observed in addition to the monomers. In toxicological tests with Daphnia magna and Photobacterium phosphoreum no significant toxicological effect was observed, neither for the monomeric intermediates nor for the oligomeric intermediates. From a risk assessment it can be concluded that there is no indication for an environmental risk when aliphatic-aromatic copolyesters of the Ecoflex-type are introduced into composting processes.
Asunto(s)
Poliésteres/metabolismo , Microbiología del Suelo , Animales , Biodegradación Ambiental , Daphnia/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Photobacterium/efectos de los fármacos , Poliésteres/efectos adversos , Medición de Riesgo , Temperatura , Pruebas de ToxicidadRESUMEN
Polymers, which undergo a controlled biological degradation by micro-organisms came to remarkable interest during the last years. Composting for instance could so be established as an alternative waste management system for parts of the plastic waste. Within this group of innovative polymer, polyesters play a predominant role, due to their potentially hydrolyzable ester bonds. While aromatic polyesters such as poly(ethylene terephthalate) exhibit excellent material properties but proved to be almost resistant to microbial attack, many aliphatic polyesters turned out to be biodegradable but lack in properties, which are important for application. To combine good material properties with biodegradability, aliphatic-aromatic copolyesters have been developed as biodegradable polymers for many years. This article reviews the attempts to combine aromatic and aliphatic structures in biodegradable plastics and work, which has been done to evaluate the degradation behaviour and environmental safety of biodegradable polyesters, containing aromatic constituents.
Asunto(s)
Bacterias/metabolismo , Poliésteres/metabolismo , Materiales Biocompatibles , Biodegradación Ambiental , Medios de Cultivo , Ambiente , Hidrólisis , Plásticos , Poliésteres/química , Temperatura , Factores de Tiempo , Administración de ResiduosRESUMEN
Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).
Asunto(s)
Bacterias Anaerobias/metabolismo , Poliésteres/metabolismo , Anaerobiosis , Bacterias Anaerobias/enzimología , Biodegradación Ambiental , Reactores Biológicos , Caproatos/metabolismo , Clostridium/crecimiento & desarrollo , Clostridium/aislamiento & purificación , Hidroxibutiratos/metabolismo , Cinética , Lactonas/metabolismoRESUMEN
Degradation of phenol and benzoic acid was studied in a fluidized-bed reactor (liquid volume 2.17 L) under nonsterile conditions with special emphasis on maximizing the flow through the reactor and investigating reactor performance at fluctuating feeds. Reactor response to substrate pulses was investigated by applying substrate square-wave inputs at a liquid flow of 1.00 L h(-1). A twofold increase of the phenol and benzoic acid feed concentrations for 2.5 h did not lead to accumulation and breakthrough. The cells were able to survive four to fivefold increases of the feed concentration for 1 h without loss of viability, although the phenol pulse lead to phenol accumulation in the reactor. Reactor performance at constantly fluctuating loads was investigated by varying the feed concentrations using sine wave functions. No accumulation of phenol or benzoic acid was observed. Influence of induction was studied using shift experiments. After 35 days of operation (369 hydrodynamic residence times) with phenol as sole substrate (carbon source) the reactor was able to mineralize benzoic acid without any adaptation or lag phase. The capability of phenol degradation, on the other hand, was lost by most cells after only 3 days operation with benzoic acid as the sole substrate. The experiments underline the importance of induction. In order to maximize the flow through the reactor, the liquid flow was increased stepwise while the feed concentrations were reduced correspondingly, keeping the volumetric conversion rates of phenol (0.24 g L(-1) h(-1)) and benzoic acid (0.17 g L(-1) h(-1)) constant. By this means, liquid flow could be increased up to 13.32 L h(-1), which was more than 20-fold higher than the maximum liquid flow achievable in a chemostat using the same conditions.
Asunto(s)
Ácido Benzoico/metabolismo , Reactores Biológicos , Biotecnología/métodos , Burkholderia cepacia/metabolismo , Fenol/metabolismo , Biotecnología/instrumentación , Dióxido de Carbono/metabolismo , Oxígeno/metabolismoRESUMEN
The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.
Asunto(s)
Alginatos/química , Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , Nitrogenasa/metabolismo , Consumo de Oxígeno , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/crecimiento & desarrollo , Azotobacter vinelandii/ultraestructura , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestructura , Reactores Biológicos , Medios de Cultivo , Ácido Glucurónico , Ácidos Hexurónicos , Fosfatos/metabolismo , Propiedades de SuperficieRESUMEN
Aggregation and precipitation are major pitfalls during bioprocessing and purification of recombinant human basic fibroblast growth factor (rh-bFGF). In order to gain high yields of the soluble protein monomer with high biological activity, an efficient downstream process was developed, focussing on the combination of expanded bed adsorption (EBA) and heparin chromatography. After expression in E. coli TG1:plambdaFGFB, cells were harvested and washed; then the rh-bFGF was released via high pressure homogenization. The high viscosity of the feedstock of about 40 mPa s, showing non-newtonian behaviour, was reduced to 2 mPa s by the addition of DNase. The homogenate (5.6 l) was loaded directly on an expanded bed column (C-50) packed with the strong cation-exchanger Streamline SP. In the eluates, histone-like (HU) protein was identified as the main protein contaminant by sequence analysis. The thermodynamics and kinetics of rh-bFGF adsorption from the whole broth protein mixture were determined in view of competition and displacement effects with host-derived proteins. Optimal binding and elution conditions were developed with knowledge of the dependence of rh-bFGF adsorption isotherms on the salt concentration to allow direct application of eluates onto Heparin HyperD. This affinity support maintained selectivity and efficiency under CIP and over a wide range of flow-rates; both is advantageous for the flexibility of the purification protocol in view of a scalable process. Remaining DNA and HU protein were separated by Heparin HyperD. The endotoxin level decreased from approximately 1,000,000 EU/ml in the whole broth to 10 EU in 3 mg bFGF per ml. The final purification protocol yields >99% pure rh-bFGF as judged from SDS-PAGE and MALDI-TOF mass spectrometry with high mitogenic activity (ED50=1-1.5 ng/ml) of the lyophilized sample. In comparison to the conventional process, the overall protein recovery rose by 15% to 65% with saving time and costs.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/aislamiento & purificación , Cromatografía de Afinidad/métodos , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Cinética , Proteínas Recombinantes/aislamiento & purificación , TermodinámicaRESUMEN
A mercury-resistant bacterial strain which is able to reduce ionic mercury to metallic mercury was used to remediate in laboratory columns mercury-containing wastewater produced during electrolytic production of chlorine. Factory effluents from several chloralkali plants in Europe were analyzed, and these effluents contained total mercury concentrations between 1.6 and 7.6 mg/liter and high chloride concentrations (up to 25 g/liter) and had pH values which were either acidic (pH 2.4) or alkaline (pH 13.0). A mercury-resistant bacterial strain, Pseudomonas putida Spi3, was isolated from polluted river sediments. Biofilms of P. putida Spi3 were grown on porous carrier material in laboratory column bioreactors. The bioreactors were continuously fed with sterile synthetic model wastewater or nonsterile, neutralized, aerated chloralkali wastewater. We found that sodium chloride concentrations up to 24 g/liter did not inhibit microbial mercury retention and that mercury concentrations up to 7 mg/liter could be treated with the bacterial biofilm with no loss of activity. When wastewater samples from three different chloralkali plants in Europe were used, levels of mercury retention efficiency between 90 and 98% were obtained. Thus, microbial mercury removal is a potential biological treatment for chloralkali electrolysis wastewater.
Asunto(s)
Mercurio/farmacocinética , Pseudomonas putida/metabolismo , Eliminación de Residuos Líquidos , Disponibilidad Biológica , Reactores Biológicos , Cloruros , Farmacorresistencia Microbiana , Electrólisis , Agua Dulce , Sedimentos Geológicos , Mercurio/farmacología , Oxidación-Reducción , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/aislamiento & purificación , Microbiología del Agua , Contaminación Química del AguaRESUMEN
1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO2, H2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH2, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a "green" polyester is a most cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.
Asunto(s)
Clostridium/metabolismo , Enterobacteriaceae/metabolismo , Fermentación , Glicoles de Propileno/metabolismo , Adenosina Trifosfato/metabolismo , Clostridium/genética , Enterobacteriaceae/genética , Ingeniería Genética , Glicerol/metabolismo , Microbiología Industrial/economía , Microbiología Industrial/métodos , Klebsiella/genética , Klebsiella/metabolismo , Oxidación-Reducción , Poliésteres/metabolismo , Ácido Pirúvico/metabolismoRESUMEN
Adsorption of pentachlorophenol (PCP) on induced cells of Mycobacterium chlorophenolicum PCP-1 and its influence on enzyme induction and PCP degradation of this strain were studied. Compared to non-induced cells, induced degrading cells had a lower adsorption capacity (q(ads)), particularly at prolonged induction and low PCP concentration. Unlike the effects of pH and biomass concentration previously reported for non-induced cells, the variation of q(ads) of induced cells was associated with changes of both the capacity and intensity constants of the Freundlich equation which was used to describe PCP adsorption on M. chlorophenolicum PCP-1. This indicated changes of cell surface properties during enzyme induction and PCP degradation. The latter was shown in turn to be affected by several parameters such as PCP concentration, pH value and induction time. Interestingly, irrespective of the pH and PCP concentration, the specific PCP degradation rate (q(t)(PCP)) at a given induction time was found to be solely a function of q(ads), revealing that adsorption capacity is an inherent key parameter for enzyme induction and PCP degradation. Based on this knowledge, a kinetic model was developed for q(t)(PCP) which used only q(ads) and induction time as variables. The model considered inhibition of PCP on both enzyme induction and enzyme activity and described the experimental data at different PCP concentrations and pH values well. q(ads) also turned out to be a useful criterion for choosing optimum induction concentration of PCP. Irrespective of pH and biomass concentration, an initial adsorption capacity of 2-3 micromol PCP/g cells was found to be optimum for enzyme induction in M. chlorophenolicum PCP-1.
Asunto(s)
Mycobacterium/enzimología , Mycobacterium/metabolismo , Pentaclorofenol/metabolismo , Adsorción , Biodegradación Ambiental , Biomasa , Reactores Biológicos , Contaminantes Ambientales/metabolismo , Inducción Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Cinética , Modelos Biológicos , Mycobacterium/efectos de los fármacos , Pentaclorofenol/farmacocinética , Pentaclorofenol/farmacologíaRESUMEN
A specially designed model reactor based on a 42-L laboratory fermentor was equipped with six stirrers (Rushton turbines) and five cylindrical disks. In this model reactor, the mixing time, Theta(90), turned out to be 13 times longer compared with the 42-L standard laboratory fermentor fitted with two Rushton turbines and four wall-fixed longitudinal baffles. To prove the suitability of the model reactor for scaledown studies of mixing-time-dependent processes, parallel exponential fed-batch cultivations were carried out with the leucine-auxotrophic strain, Corynebacterium glutamicum DSM 5715, serving as a microbial test system. L‐Leucine, the process-limiting substrate, was fed onto the liquid surface of both reactors. Cultivations were conducted using the same inoculum material and equal oxygen supply. The model reactor showed reduced sugar consumption (-14%), reduced ammonium consumption (-19%), and reduced biomass formation (-7%), which resulted in a decrease in L-lysine formation (-12%). These findings were reflected in less specific enzyme activity, which was determined for citrate synthase (CS), phosphoenolpyruvate carboxylase (PEP-C), and aspartate kinase (AK). The reduced specific activity of CS correlated with lower CO(2) evolution (-36%) during cultivation. The model reactor represents a valuable tool to simulate the conditions of poor mixing and inhomogeneous substrate distribution in bioreactors of industrial scale. Copyright 1999 John Wiley & Sons, Inc.
RESUMEN
Batch and fed batch cultivations of Xanthomonas campestris with varying amounts of the N source (NH4Cl) yield xanthan polymers which differ in pyruvate content. Analyzing the rheological properties of the xanthans produced exhibits that it is not only the molecular mass but also the pyruvate content that affects the viscosity of xanthan solutions. However, there is obviously no simple functional dependency but rather a step increase in viscosity when going from below 2 to above 3 wt % of pyruvate.
RESUMEN
Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Modelos Biológicos , Animales , Biotecnología , Recuento de Células , Muerte Celular , División Celular , Línea Celular , PerfusiónRESUMEN
A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations.
Asunto(s)
Reactores Biológicos , Tetrahymena thermophila/crecimiento & desarrollo , Animales , Biotecnología , Medios de Cultivo/química , Endopeptidasas/metabolismo , Fermentación , Tetrahymena thermophila/enzimologíaRESUMEN
Chromatography in stable expanded beds enables proteins to be recovered directly from cultivations of microorganisms or cells and preparations of disrupted cells, without the need for prior removal of suspended solids. The general performance of an expanded bed is comparable to a packed bed owing to reduced mixing of the adsorbent particles in the column. However, optimal operating conditions are more restricted than in a packed bed due to the dependence of bed expansion on the size and density of the adsorbent particles as well as the viscosity and density of the feedstock. The feedstock composition may become the most limiting restriction owing to interactions of adsorbent particles with cell surfaces, DNA and other substances, leading to their aggregation and consequently to bed instabilities and channeling. Despite these difficulties, expanded-bed chromatography has found widespread applications in the large scale purification of proteins from mammalian cell and microbial feedstocks in industrial bioprocessing. The basics and implementation of expanded-bed chromatography, its advantages as well as problems encountered in the use of this technique for the direct extraction of proteins from unclarified feedstocks are addressed.
Asunto(s)
Cromatografía/métodos , Proteínas/aislamiento & purificación , Adsorción , Alimentación Animal/análisis , Cromatografía/instrumentación , Tamaño de la PartículaRESUMEN
Material and degree of reductance balance equations are used to estimate the rates of oxygen uptake and carbon dioxide evolution of animal cell cultures. Lumped compositions, molecular weight and reductance degree of cellular protein, monoclonal antibody, biomass and amino acid consumption (excluding glutamine and alanine) are found to be relatively constant for different hybridoma cell lines and may be used as regularities. The calculated rates of oxygen uptake and carbon dioxide evolution agree well with experimental values of several different cultures reported in the literature. This simple method gives the same results as calculated on the basis of a detailed metabolic reaction network.
RESUMEN
Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this p range 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed.
Asunto(s)
Butileno Glicoles/química , Glicerol/metabolismo , Klebsiella pneumoniae/metabolismo , Glicoles de Propileno/química , Ácido Acético/química , Acetoína/química , Cromatografía de Gases , Etanol/química , Fermentación , Glicerol/análisis , Concentración de Iones de Hidrógeno , Cinética , Ácido Láctico/análisisRESUMEN
Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120,000 in protein-free solutions and 40 to 33,000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.