bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrafiltration membranes for chemical bonding of urease
Authors: Schmidt‐Steffen Alfred , Staude Eberhard
Published: December 2004

Asymmetric ultrafiltration membranes suitable for covalent bonding of urease can be prepared from membranes based upon polyamide or polysulfone. Because the original membrane polymers are not chemically reactive, they have to be converted in such a way that known reactions for enzyme fixation can be used such as the diazo, the acyl‐acid, the carbodiimide, and the methylbromide reaction. The enzyme was fixed within the porous substructure of the membrane. The amount of enzyme immobilized at the membrane dense skin was found to be negligible. The kinetics can be described according to the Michaelis–Menten model. Compared to the native urease, the activity of the membrane‐bonded enzyme was very low. The reasons can be sought in the transmembrane transport and in the fixation procedure as well.
Development of a multienzyme reactor for dopamine synthesis: I. Enzymology and kinetics
Authors: Anderson William A. , Moo‐Young Murray , Legge Raymond L.
Published: December 2004

The enzymology and kinetics of tyrosine phenol lyase (TPL) from Erwinia herbicola, and tyrosine decarboxylase (TDC) from Streptococcus faecalis have been investigated for potential use in a coimmobilized multienzyme biocatalytic system for the production of dopamine. In this multienzyme biotransformation using whole cells optimized for each of the respective enzymes, TPL catalyzes the production of 3,4‐dihydroxyphenyl‐L‐alanine (L‐dopa) from catechol, pyruvate, and ammonium, and this is subsequently decarboxylated by TDC to produce dopamine. Performing the reactions simultaneously, thereby removing L‐dopa, is one option for overcoming the TPL equilibrium constraints. The enzymes have different optimal pH values, so the reaction kinetics at a compromise pH of 7.1, where both enzymes could be operated simultaneously, were investigated. For the concentration range investigated, TPL followed pseudo‐first‐order kinetics with respect to catechol, pyruvate, and ammonium. TDC exhibited significant product inhibition as well as inhibition by combinations of catechol and pyruvate.
Protein extraction and activity in reverse micelles of a nonionic detergent
Authors: Ayala Guadalupe A. , Kamat Sanjay , Beckman Eric J. , Russell Alan J.
Published: December 2004

We describe, for the first time, the ability of a polyoxyethylene sorbitan trioleate‐isopropanol microemulsion in hexane to solubilize pure proteins. The dependences of cytochrome c extraction and buffer solubilization by the reverse micellar system on ionic strength of the aqueous phase, detergent concentration, and cosurfactant concentration result in increased extraction. In addition, subtilisin (a serine protease) is shown to be active in this microemulsion. Further the activity of the enzyme can be regulated by the water content of the micelles, enabling control of enzyme activity by “solvent engineering.”
Analysis of mammalian viable cell biomass based on cellular ATP
Authors: Sonderhoff Stefan A. , Kilburn Douglas G. , Piret James M.
Published: December 2004

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin–luciferase assay was compared with trypan blue‐stained hemocytometer counts. The cell‐specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell‐specific ATP than nonviable cells. Cell‐specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.
Oxygen transfer modeling and simulations for an industrial continuous airlift fermentor
Authors: Pigache S. , Dhoms P. , Trystram G.
Published: December 2004

This article deals with the modeling of the oxygen transfer in an industrial airlift fermentor used for lactic yeast production on whey substrates. The purpose of this study was to improve the understanding of the interactions among the various parameters that govern the oxygen transfer phenomena in this type of fermentor. The reliability of the proposed model is demonstrated. The results of the investigations have been put into practice on the industrial scale and have contributed to monitor better the fermentation process. The model was also used to develop new ways of industrial fermentor design.
Production of a discrete, heterogeneous population of β‐galactosidase polypeptides using baculovirus expression vectors
Authors: Licari P. , Bailey J. E.
Published: December 2004

Gel electrophoresis analysis of immunoprecipitated β‐galactosidase and polyhedrin‐β‐galactosidase expressed in Spodoptera frugiperda cells infected with recombinant Autograph californica nuclear polyhedrosis virus revealed the existence of a population of discrete β‐galactosidase polypeptides. Several of the polypeptides observed in the fusion protein expression experiments exhibit a consistent pattern of slightly greater molecular weight when compared to the nonfusion β‐galactosidase that is compatible with the hypothesis that these fusion protein fragments retain the N‐terminal polyhedrin residues. Pulse‐chase experiments showed that overall β‐galactosidase degradation occurred at a negligible rate compared to the synthesis rate at 96 h postinfection, yet the fragments are observed for short pulse times. Degradation of several different β‐galactosidase polypeptides was observed 24 h postinfection. Ribonucleic acid hybridization analysis of lacZ transcripts shows significant heterogeneity that may result from premature transcription termination. Although a proteolytic origin cannot be excluded, the data assembled suggest that premature termination of transcription or translation is the likely cause for the heterogeneous population of immunoreactive peptides observed. Many discrete forms of β‐galactosidase polypeptides were also observed in studies with Escherichia coli, indicating that production of these heterogeneous forms is not a consequence of heterologous expression of the enzyme.
Classification and measurement of fungal pellets by automated image analysis
Authors: Cox P. W. , Thomas C. R.
Published: December 2004

An automated image analysis method for classifying and measuring pellets of filamentous fungi growing in submerged fermentations has been developed. The method discriminates between pelleted mycelial growth and loose aggregates of dispersed hyphae. Pellets are classified into smooth and hairy types. In both cases, the core of the pellet is identified and its shape and size characterized. For hairy pellets the annular region is also characterized. The method was tested on pellets of Aspergillus niger ATCC 11414 grown in a defined medium in shake flasks. This rapid method makes practical extensive studies on the morphology of pellets in submerged fermentations and the influence of fermentation conditions on that morphology.
Hydrolysis of butteroil by immobilized lipase using a hollow‐fiber reactor: Part II. Uniresponse kinetic studies
Authors: Malcata F. Xavier , Hill Charles G. , Amundson Clyde H.
Published: December 2004

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the hydrolysis of the glycerides of melted butterfat at 40°C and pH 7.0. Mcllvane buffer was pumped through the lumen and melted butterfat was pumped courrently through the shell side of a shell‐and‐tube reactor. Nonlinear regression methods were employed to determine the kinetic parameters of three nested rate expressions derived from a Ping Pong Bi Bi enzymatic mechanism coupled with three nested rate expressions for the thermal deactivation of the enzyme. For the reaction conditions used in this research, a four‐parameter rate expression (which includes a two‐parameter deactivation rate expression and a two‐parameter hydrolysis rate expression) is sufficient to model the overall release of free fatty acids from the triglycerides of butterfat as a function of space time and time elapsed after immobilization. At a space time of 3.7 h immediately after immobilization of lipase, 50% of the fatty acid residues esterified in the sn‐1,3 positions of the triglycerides can be released in the hollow‐fiber reactor.
The use of Fab‐masking antigens to enhance the activity of immobilized antibodies
Authors: Velander William H. , Subramanian Anuradha , Madurawe Rapti D. , Orthner Carolyn L.
Published: December 2004

This study demonstrates that masking the Feb regions of a monoclonal antibody (Mab) with synthetic antigens prior to covalent immobilization efficiency. Water‐soluble adducts of poly(2‐methyloxazoline) polymers and a syntheticpeptide epitope for the Mab were constructed. These synthetic antigens are referred to as Fab‐masking antigents (FMAs). The antibody used in this study is a Ca2+‐dependent murine monoclonal lgG directed against the plasma protein, human protein C (hPC). The FMAs were pre‐equilibrated with Mab in the presence of calcium prior to immobilization and were then removed by EDTA, which destabilized the FMA‐Mab complexes. The antigen binding efficiency and accessibility of the Fab domain of the immobilized antibody was significantly increased for Mab immobilized in the presence of FMA relative to those Mab immobilized without FMA. The increase in binding efficiency was most pronounced for the largest FMA employed. No appreciable differences were detected in the avidity of hPC‐Mab complexes formed by immunosorbents produced by either masked or unmaked antibody. These results provide evidence that orientgation may play an important role in the binding activity of immobilized antibodies.
Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis
Authors: Mueller Robert F. , Characklis William G. , Jones Warren L. , Sears John T.
Published: December 2004

The processes leading to bacterial colonization on solidwater interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless‐steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m−2. The surface roughness varied among the substrata from 0.002 μm (for silicon) to 0.015 μm (for copper). Surface free energies varied from 25.1 dynes cm−1 for silicon to 31.2 dynes cm−1 for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.

The adsorption rate coefficient varried by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell‐containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.
Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures
Authors: Srinivasan Venkatesh , Ryu D. D. Y.
Published: December 2004

The activities of the biosynthetic enzmes phenylalanine ammonia lyase (PAL) and 3‐hydroxy‐3‐methylglutaryl‐CoA‐reductase (HMGR) were measured in cells transferred from growth to production medium in a two‐stage batch culture. It was found that both these enzymes showed transient increases, PAL (three‐ to fourfold) and HMGR (two‐ to four‐fold), at or near the point of exhaustion of nitrogen source (NO3). Production of shikonin derivatives also started at this time. The addition of excess nitrate to the medium shortly before nitrate exhaustion (days 6–8) markedly reduced the final product yield (by 70–80%) while addition of excess nitrate in the later stationary growth phase (days 14–16) had no significant effect. When the production rate of shikonin derivatives was correlated with PAL activity, it was observed that production rate is very low (less than 1 mg/L · day) at low levels of PAL activity (below 0.1 unit/mg protein). Once a threshold level of PAL activity (about 0.15 unit/mg protein) is reached, the biosynthetic rate of shikonin derivatives increases. Such a relationship could not be deduced for HMGR activity. It was concluded that the production of shikonin derivatives may be limited at the phenylalanine deaminating step at low levels of PAL activity.
Formation of polyol–fatty acid esters by lipases in reverse micellar media
Authors: Hayes Douglas G. , Gulari Erdogan
Published: December 2004

The synthesis of polyol–fatty acid esters has strong implications in such industries as foods, cosmetics, and polymers. We have investigated these esterification reactions employing the polyols ethylene glycol, 2‐monoglyceride, and sugars and their dervatives with the biocatalyst lipase in water/AOT/isooctane reverse micellar media. For the first reaction, 50–60% conversion was achieved and product selectivity toward the monoester over the diester shown possible by employing lipase from Rhizopus delemar. A simple kinetic model based on the formation of acyl‐enzyme intermediate accurately predicted the effect of polyol concentration but not the effect of fatty acid or water concentration probably due to the model exclusion of paritioning effects. The success of this reaction in reverse micellar media is due greatly to its capacity to solubilize large quantities of glycol despite the media's overall hydrophobicity. The second reaction, investigated for its potential for production of “mixed” glycerides, also achieved about 50% conversion but had only a small portion of triglyceride in its product distribution. Also, isomerization of the 2‐monoglyceride to 1‐monoglyceride, followed by hydrolysis of the latter, unfortunately occurred to a significant extent. Attempts at esterification with hexoses and their derivatives such as glucose and mannitol produced no convesion.
Anomeric specificity of glucose uptake systems in Lactococcus cremoris, Escherichia coli, and Saccharomyces cerevisiae: Mechanism, kinetics, and lmplications
Authors: Benthin Stig , Nielsen Jens , Villadsen John
Published: December 2004

The mechanism and kinetics of the glucose uptake systems of three representative microorganisms are studied during cultivation in a chemostat. The three microorganisms are Lactococcus cremoris, Escherichia coli, and Saccharomyces cervisiae. Two models describing respectively competitive and independent uptake of the two glucose anomers are tested on experimental data where α‐ and β‐glucose are determined by flow injection analysis after pulse addition of the pure anomers to a chemostat. The very accurate experimental results are used to give a convincingly clear model discrimination for all three microorganisms. The uptake of glucose by S. cervisiae occurs by a competitive mechanism with preference for α‐glucose (Kα = 32 mg/L and Kβ = 48 mg/L). Surprisingly, the glucose uptake by the two bacteria is shown to be mediated by anomer specific transport systems with no competitive inhibition from the other glucose anomer. This novel finding has not been described in the literature on the phosphotransferase system. In L. cremoris the relative uptake rates of the glucose anomers match the equilibrium composition exactly (36% α‐glucose). In E. coli the relative uptake rate of α‐glucose at glucose unlimited growth is 26%, which means preference for β‐glucose. However, the saturation constants of the two sites in E. coli are Kα = 2 mg/L and Kα = 15 mg/L, and a preference for α‐glucose is exhibited at very low glucose concentrations. The results are of considerable improtance in relation to enzyme based on‐line measurements during fermentations as well as to the modeling of glucose limited growth and product formation.
Incapability of Gluconobacter oxydans to produce tartaric acid
Authors: Klasen Ralf , Bringer‐Meyer Staphanie , Sahm Hermann
Published: December 2004

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that trataric acid could only be produced in a medium containing vanadate (NH4VO3). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5‐ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5‐ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L‐(+)‐type. Oxalic acid was formed from 5‐ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.
High oxygen transfer rate in a new aeration system using hollow fiber membrane
Authors: Matsuoka Hiroshi , Fukada Shouhei , Toda Kiyoshi
Published: December 2004

A new type of bubble aeration column called a hollow fiber membrane (HFM) aeration column was proposed, which was featured in the use of hollow fiber membranes and gave a high bubble density in the column. The value of kLa was increased by modifying the membrane surface for making the pore size smaller. The Sauter mean diameter of bubbles (Dvs) was 2.0 ± 0.1 mm in the range of the superficial gas velocity from 0.02 m s−1 to 0.065 m s−1, while that obtained for the bubbles near the membrane was 811 μm at the superficial gas velocity of 4.0 × 10−4 m s−1. The difference was ascribed to the effect of coalescence of bubbles. The value of KLa increased in proportion to the superficial gas velocity up to 0.02 m s−1, and was almost constant above 0.03 m s−1. The maximum value of kLa, 2.5 s−1, was higher than those of the other aeration columns reported previously. The pneumatic power consumption per unit liquid volume (Pv) for obtaining the same kLa was the smallest in the HFM aeration columns. Pv, for obtaining the same interfacial area of bubbles per liquid volume, was also lower than those for other types of aeration columns. It was suggested from the measurement of bubble diameter that the larger interfacial area generated in the HFM aeration column ascribes to the larger gas holdup than the smaller Dvs. © 1992 John Wiley & Sons, Inc.
Direct measurement of the yield stress of filamentous fermentation broths with the rotating vane technique
Authors: Leong‐Poi Lisa , Allen D. Grant
Published: December 2004

The existence of a yield stress in filamentous fermentation broths has important transport phenomena implications in the design and operation of bioreactors. In this study, the constant shear rate vane method was assessed for directly measuring the yield stress of filamentous Aspergillus niger fermentation broths, as well as model fluids (ketchup, yogurt, and pulp suspensions). The method involved rotating 4‐, 6‐, and 8‐bladed vanes (7.2 cm ≤ height ≤ 15 cm; 4.0 cm ≤ dia. ≤ 6 cm) at speeds of 0.01 to 0.64 rpm in the fluid and plotting the torque as a function of time. Based on visual observations, the consistency of the results with vane type and speed and comparison with previous work on nonbiological samples, it was concluded that the method is an effective and consistent technique for yield stress measurements on filamentous fermentation broths. Based on comparisons with concentric cylinder viscometer results, it was also concluded that the value determined via the vane method was a “static” yield stress (values of up to 28 Pa) which was much greater than the extrapolated (or “dynamic”) yield stress determined via the concentric cylinder viscometer. © 1992 John Wiley & Sons, Inc.
Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition
Authors: Crumbliss A. L. , Perine S. C. , Stonehuerner J. , Tubergen K. R. , Zhao Junguo , Henkens R. W. , O'Daly J. P.
Published: December 2004

Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K′M). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. © 1992 John Wiley & Sons, Inc.
Effect of reducing agents in an aerobic amino acid fermentation
Authors: Kwong Simon C. W. , Rao Govind
Published: December 2004

This study focuses on the effects of the reducing agents, dithiothreitol (DTT) and glutathione (GSH), on amino acid production in aerobically growing Corynebacterium glutamicum. The problem of reducing agent addition affecting the dissolved oxygen level was solved by positioning the culture at a high dissolved oxygen level and feeding the reducing agent into the fermentor. We show that it is possible to lower the redox potential even in a highly aerobic environment. The addition of DTT to the fermentation during the growth phase caused a significant increase in specific amino acid production rate and total amino acids produced, as compared with a control. In contrast, GSH had an inhibitory effect. © 1992 John Wiley & Sons, Inc.
Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors
Authors: van Gulik W. M. , ten Hoopen H. J. G. , Heijnen J. J.
Published: December 2004

Plant cell suspension cultures of Catharanthus roseus and Nicotiana tabacum were grown in stirred tank bioreactors operated in batch and continuous mode. The stoichiometry of growth of both species in steady‐state glucose limited chemostats was studied at a range of different dilution rates. A linear relation was applied to describe specific glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate. Specific respiration deviated greatly from the linear relation. An unstructured mathematical model, based on the observed stoichiometry in the glucose limited chemostats, was applied to describe the growth in batch culture. From a comparison between the observed growth pattern in batch fermentors and computer simulations it appeared that the stoichiometry of growth of the C. roseus culture was different under steady‐state and dynamic conditions. It was concluded that a mathematical model for the growth of suspension culture plant cells in which the biomass is considered to be a single compound with an average chemical composition is of limited value because large changes in the conmposition of the biomass may occur. © 1992 John Wiley & Sons, Inc.
Mechanism of insulin aggregation and stabilization in agitated aqueous solutions
Authors: Sluzky Victoria , Klibanov Alexander M. , Langer Robert
Published: December 2004

Undesirable aggregation of aqueous insulin solutions remains a serious obstacle in the development of alternative methods of diabetes therapy. We investigated the fundamental nature of the aggregation mechanism and proposed stabilization strategies based on a mathematical model for the reaction scheme. Insulin aggregation kinetics in the presence of solid‐liquid and air–liquid interfaces were monitored using UV spectroscopy and quasielastic light scattering (QELS). Experimental observations were consistent with our model of monomer denaturation at hydrophobic surfaces followed by the formation of stable intermediate species which facilitated subsequent macroaggregation. The model was used to predict qualitative trends in insulin aggregation behavior, to propose stabilization strategies, and to elucidate mechanisms of stabilization. In the absence of additives, insulin solutions aggregated completely (more than 95% of the soluble protein lost) within 24 h; with sugarbased nonionic detergents, no detectable loss occurred for more than 6 weeks. © 1992 John Wiley & Sons, Inc.
Investigation of reduced serum and serum‐free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)
Authors: Zhang Junli , Kalogerakis Nicolas , Behie Leo A. , Iatrou Kostas
Published: December 2004

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum‐free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum‐free medium (EX‐CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL‐41 + 2% fetal bovine serum (FBS), SF‐900, and a serum‐free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum‐supplemented medium and a serum‐free medium (EX‐CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum‐supplemented medium. © 1992 John Wiley & Sons, Inc.
Nutrient‐dependent selection of morphological mutants ofFusarium graminearumA3/5 isolated from long‐term continuous flow cultures
Authors: Wiebe Marilyn G. , Robson Geoffrey D. , Cunliffe Bryan , Trinci Anthony P. J. , Oliver Stephen G.
Published: December 2004

Highly branched (colonial) mutants (MC1‐1‐, CC1‐1, and C106) ofFusarium graminearumA3/5 were each grown with the parental strain (A3/5) in continuous flow cultures at high and low dilution rates using a variety of nutrient limitations. MC1‐1 replaced A3/5 in all nutrient‐limited cultures tested (glucose‐, Mg2+‐, ammonium‐, and sulphate‐limited cultures), suggesting that it has a higher maximum specific growh rate than A3/5. Compared with A3/5, C106 was positively selected for in Mg2+‐limited cultures and its selection coefficient was higher at low than at high dilution rates, suggesting that, compared with A3/5, it has a reduced saturation constant (Ks) for Mg2+. However, in batch culture, C106 and A3/5 had the same (15 μM) appaentKsvalue for Mg2+. C106 was replaced (negative selection coefficient) by A3/5 in gluose‐, ammonium‐, and phsophate‐limited continuous flow cultures, but was neither at an advantage nor a disadvantage (i.e., it behaved as a neutral mutation) in sulphate‐limited cultures. CC1‐1 replaced A3/5 when they were grown together in glucose‐, maltose‐, or ribose‐limited continuous flow cultures, but not in fructose‐, xylose‐, ammonium‐, or phsophate‐limited cultures. Because A3/5 and CC1‐1 had similarKmvalues (30 μM) for glucose, and because the selective advantage of CC1‐1 was maintained in maltose‐limited cultures (maltose was not hydrolyzed extracellularly), it was concluded that the selective advantage of CC1‐1 did not result from it having a lowerKsfor glucose than the parental strain. Rather, the data suggested that the activity of phosphoketopentoepimerase may be altered by the CC1‐1 mutation. © 1992 John Wiley & Sons, Inc.
Optimizing the sample size and the retention parameters to achieve maximum production rates for enantiomers in chiral chromatography
Authors: Jacobson Stephen C. , Felinger Attila , Guiochon Georges
Published: December 2004

The optimum experimental conditions (sample size and mobile phase composition) are calculated for maximum production rate of either one of two enantiomers contained in feeds of different compositions (1/1, 1/10, and 10/1). The products are obtained at 99% purity. The calculations use the equilibrium‐dispersive model of chromatography and the equilibrium isotherms determined experimentally from the rear, diffuse boundary of overloaded elution profiles. The production rate measured experimentally under the optimum conditions calculated agree with 4% of the calculated values. There is an optimum value for the retention factor which is higher than predicted by a model assuming constant separation factor, because both separation factor and retention decrease with increasing organic solvent concentration in the mobile phase. © 1992 John Wiley & Sons, Inc.
Oxygen uptake rates in cultured rat hepatocytes
Authors: Rotem Avi , Toner Menmet , Tompkins Ronald G. , Yarmush Martin L.
Published: December 2004

One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large‐scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long‐term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 ± 0.57 pmol/sec/μg DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 ± 1.5 pmol/sec/μg DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage‐dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. © 1992 John Wiley & Sons, Inc.
Influence of oxygen tension, biomass concentration, and specific growth rate on the rheological properties of a filamentous fermentation broth
Authors: Olsvik E. S. , Kristiansen B.
Published: December 2004

The influence of oxygen tension, biomass concentration, and specific growth rate on the rheological properties of an Aspergillus niger fermentation broth was investigated by growing the fungus in continuous culture. The rheological properties were measured on‐line using an impeller rheometer system. The effect of the specific growth rate on broth rheology was strongly influenced by the dissolved oxygen, (DO) concentration in the broth. At DO concentrations above 10% of saturation, K increased with the dilution rate, and at DO concentrations below 10% of saturation, K decreased with increasing dilution rate. The largest influence of a change in the DO concentration on the viscosity of the broth was found at the lowest growth rates and the lowest DO concentrations. K/x, a term that gives a simple description of the structure or the morphology of the culture, was found to increase with biomass concentration. © 1992 John Wiley & Sons, Inc.
Purification of recombinant protein A by aqueous two‐phase extraction integrated with affinity precipitation
Authors: Kamihira Masamichi , Kaul Rajni , Mattiasson Bo
Published: December 2004

Aqueous two‐phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two‐phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two‐phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26‐fold by thid method. The IgG‐Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial‐scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two‐phase method and affinity precipitation method. © 1992 John Wiley & Sons, Inc.
Mineralization of biphenyl and PCBs by the white rot fungus Phanerochaete chrysosporium
Authors: Thomas Daniel R. , Carswell Kathleen S. , Georgiou George
Published: December 2004

The white rot fungus Phanerochaete chrysosporium is unique in its ability to totally degrade a wide variety of recalcitrant pollutants. We have investigated the degradation of biphenyl and two model chlorinated biphenyls, 2,2′,4,4′‐tetrachlorobiphenyl and 2‐chlorobiphenyl by suspended cultures of P. chrysosporium grown under conditions that maximize the synthesis of lignin‐oxidizing enzymes. Radiolabeled biphenyl and 2′‐chlorobiphenyl added to cultures at concentrations in the range 260 nM to 8.8 μM were degraded extensively to CO2 within 30 days. In addition, from 40% to 60% of the recovered radioactivity was found in water‐soluble compounds. A correlation between the rate of degradation and the synthesis of ligninases or Mn‐dependent peroxidases could not be observed, indicating that yet unknown enzymatic system may be resonsible for the initial oxidation of PCBs. The more heavily chlorinated PCB congener, 2,2′,4,4′‐tetrachlorobiphenyl was converted to CO2 less readily; approximately 9% and 0.9% mineralization was observed in cultures incubated with 40 nM and 5.3 μM, respectively. Overall, our results indicate that P. chrysosporium is a promising organism for the treatment of wastes contaminatd with lightly and moderately chlorinated PCBs. © 1992 John Wiley & Sons, Inc.
Biodegradation of an inhibitory nongrowth substrate (nitroglycerin) in batch reactors
Authors: Pesari Harish , Grasso Domenic
Published: December 2004

Biodegradation of nitroglycerin (NG), an inhibitory, nongrowth substrate present in a multicomponent munition wastewater, was investigated in a pilot‐scale batch reactor operated with both aerobic and anoxic cycles. A mixed culture was initially acclimated by gradual introduction of NG into influent and subsequently exposed to actual NG‐laden production wastewater. System performance revealed that NG was amenable to aerobic biodegradation without adverse impact on removal efficiencies of other pollutants. Temporal NG concentration profiles indicated that an influent concentration of approximately 200 mg/L of NG was reduced to below detection limits in less than 5 h of aeration with no appreciable (<4%) biosorption. Failure of NG‐acclimated cultures to utilize NG as a sole carbon source in bench‐scale reactors suggested that NG behaved as a non‐growth substrate and its degradation possibly occurred by cometabolism. Ethyl acetate present in the waste stream was an adequate growth substrate in terms of both biological and physicochemical properties. High concentrations of NO3‐N, produced as a result of aerobic degradation of NG and other nitrogenous compounds of the waste, were treated in an anoxic phase. Approximately 95 mg/L of NO3‐N was denitrified to below detection limits in 5 h of anoxia without the addition of external carbon sources. Two SRB cycle schemes with different static‐fill times exhibited significant differences in treatment efficiencies. © 1993 John Wiley & Sons, Inc.
Cultivation of mammalian cells as aggregates in bioreactors: Effect of calcium concentration of spatial distribution of viability
Authors: Peshwa Madhusudan V. , Kyung Yun‐Seung , McClure Don B. , Hu Wei‐Shou
Published: December 2004

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 μM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large‐scale operation. © 1993 John Wiley & Sons, Inc.
Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5α(pMJR1750)
Authors: Huang Ching‐Tsan , Peretti Steven W. , Bryers James D.
Published: December 2004

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5α(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5α(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid‐bearing cells in suspended batch culture dropped from 0.45 h−1 to 0.35 h−1 and the β‐galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h−1, about 36% of that without IPTG, and the β‐galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.

In biofilm cultures, both plasmid‐bearing and plasmid‐free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm‐bound E. coli DH5β(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid‐bearing cells in biofilm dropped dramatically while plasmid‐free cell numbers maintained unaffected. The β‐galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. © 1993 John Wiley & Sons, Inc.
Defined media optimization for growth of recombinant Escherichia coli X90
Authors: Yee L. , Blanch H. W.
Published: December 2004

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose‐limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h−1 for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose‐limited chemostat culture. © 1993 John Wiley & Sons, Inc.
In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin
Authors: Asakura Tetsuo , Sakaguchi Ryuji , Demura Makoto , Manabe Takuro , Uyma Atsuo , Ogawa Katsuaki , Osanai Minoru
Published: December 2004

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient 15N isotope labeling of silk fibroin, which is required for 15N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. © 1993 John Wiley & Sons, Inc.
Biological sulfuric acid transformation: Reactor design and process optimization
Authors: Stucki Gerhard , Hanselmann Kurt W. , Hürzeler Richard A.
Published: December 2004

As an alternative to the current disposal technologies for waste sulfuric acid, a new combination of recycling processes was developed. The strong acid (H2SO4) is biologically converted with the weak acid (CH3COOH) into two volatile weak acids (H2S, H2CO3) by sulfate‐reducing bacteria. The transformation is possible without prior neutralization of the sulfuric acid. The microbially mediated transformation can be followed by physiochemical processes for the further conversion of the H2S.

The reduction of sulfate to H2S is carried out under carbon‐limited conditions at pH 7.5 to 8.5. A fixed‐bed biofilm column reactor is used in conjunction with a separate gas‐stripping column which was installed in the recycle stream. Sulfate, total sulfide, and the carbon substrate (in most cases acetate) were determined quantitatively. H2S and CO2 are continually removed by stripping with N2. Optimal removal is achieved under pH conditions which are adjusted to values below the pKa‐values of the acids. The H2S concentration in the stripped gas was 2% to 8% (v/v) if H2SO4 and CH3COOH are fed to the recycle stream just before the stripping column.

Microbiol conversion rates of 65 g of sulfate reduced per liter of bioreactor volume per day are achieved and bacterial conversion efficiencies for sulfate of more than 95% can be maintained if the concentration of undissociated H2S is kept below 40 to 50 mg/L. Porous glass spheres, lava beads, and polyurethane pellets are useful matrices for the attachment of the bacterial biomass. Theoretical aspects and the dependence of the overall conversion performance on selected process parameters are illustrated in the Appendix to this article. © 1993 John Wiley & Sons, Inc.
Cell Culture conditions determine the enhancement of specific monoclonal antibody productivity of calcium alginate‐entrapped S3H5/γ2bA2 hybridoma cells
Authors: Lee Gyun Min , Chuck Alice S. , Palsson Bernhard O.
Published: December 2004

Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (qMAb) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the qMAb with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/ϒ2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T‐ and spinner flasks in order to determine whether cultivation methods (T‐ and spinner flasks) and bead size influence the qMAb Free‐suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the qMab of the entrapped cells are reversible.

The cultivation methods was found to influence significantly the qMAb of the entrapped cells. When the entrapped cells in 1‐mn diameter beads were cultivated in T‐flasks, the qMAb was not increased by 200% as previously observed in an entrapped cell culture using 1‐mm‐diameter alginate beads in spinner flasks. The qMAb of the entrapped cell was approximately 58% higher than that of the free‐suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1‐ to 3‐mm diameter did not significantly influence the qMAb, regardless of cultivations methods. The changes in qMAb of an entrapped cells were reversible. When the free‐suspended cells recovered from the T‐ and spinner flasks were sub‐cultured in T‐ and spinner flasks enhanced qMAb of the entrapped cells in both cases decreased to the level of the free‐suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the qMAb. © 1993 John Wiley & Sons, Inc.
Effect of dilution rate on growth, productivity, cell cycle and size, and shear sensitivity of a hybridoma cell in a continuous culture
Authors: Martens D. E. , de Gooijer C. D. , van der Velden‐de Groot C. A. M. , Beuvery E. C. , Tramper J.
Published: December 2004

To study the effects of the growth rate of the hybridoma cell Mn12 on productivity, cell cycle, cell size, and shear sensitivity, six continuous cultures were run at dilution rate of 0.011, 0.021, 0.023, 0.030, 0.042, and 0.058 h−1. This particular hybridoma cell appeared to be unstable in continuous culture with respect to specific productivity, as a sudden drop occurred after about 30 generations in continuous culture, accompanied by the appearance of two populations with respect to the cytoplasmic lgG content. The specific productivity increased with increasing growth rate. The shear sensitivity of the cell, as measured in a small air‐lift loop reactor, increased with increasing growth rate. The mean relative cell size, as determined with a flow cytometer, increased with increasing growth rates. Furthermore, the fraction of cells in the S phase increased, and the fraction of cells in the G1/G0 phase decreased with increasing growth rates. © 1993 John Wiley & Sons, Inc.