bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid purification of DesPro(2)‐Val15‐Leu17‐aprotinin from the culture broth of a recombinant Saccharomyces cerevisiae
Authors: Barthel Thomas , Kula Maria‐Regina
Published: December 2004

A rapid two‐step procedure has been developed for the purification of Despro(2)‐Val15‐Leu17‐aprotinin from the culture supernatant of a recombinant yeast by affinity and ion‐exchange chromatography. DesPro(2)‐Val15‐Leu17‐aprotinin was purified to homogeneity, as demonstrated by dodecylsulfate gel electrophoresis and analysis of the N‐terminal amino acid sequence. © 1993 John Wiley & Sons, Inc.
High tolerance of methanogens in granular sludge to oxygen
Authors: Kato Mario T. , Field Jim A. , Lettinga Gatze
Published: December 2004

This research assessed the effect of oxygen exposure on the methanogenic activity of anaerobic granular sludges. The toxicity of oxygen to acetoclastic methanogens in five different anaerobic granular sludges was determined in serum flasks with effective gas‐to‐liquid volumes of 4.65 to 1. The amount of oxygen that caused 50% inhibition of the methanogenic activity after 3 days of exposure ranged from 7% to 41% oxygen in the head space. These results indicate that methanogens located in granular sludge have a high tolerance for oxygen. The most important factor contributing to the tolerance was the oxygen consumption by facultative bacteria metabolizing biodegradable substrates. Uptake of oxygen by these bacteria creates anaerobic microenvironments where the methanogenic bacteria are protected. The results also indicate that methanogens in sludge consortia still have some tolerance to oxygen, even in the absence of facultative substrate for oxygen respiration. © 1993 John Wiley & Sons, Inc.
Modeling the regulation of bacterial genes producing proteins that strongly influence growth
Authors: Axe Douglas D. , Bailey James E.
Published: December 2004

A theoretical method for comparing the performance of rival models of bacterial genetic regulation is presented. The particukar difficulties involved in describing the regulated synthesis of proteins that strongly influence cell growth are identiied, and the method is specifically designed to treat such cases. The method employs a mathematical description of intrinsic perturbations occurring during exponential growth to test the performance of regulatory models. Specific models of transcriptional and translational regulation are inserted into a general gene‐expression framework in order to determine their control responses. Applying thhis approach to examine the regulation of RNA polymerase synthesis in Eschericia coli provides support for the hypothesis that rpoB translation is regulated by cooperative binding of multiple RNA polymerase molecules to the mRNA. The framework is of a sufficiently general form that the method can be used to study mechanisms involved in controlling synthesis of any bacterial protein. © 1994 John Wiley & Sons, Inc.
Kinetic analysis of microbial sulfate reduction by desulfovibrio desulfuricans in an anaerobic upflow porous media biofilm reactor
Authors: Chen Ching‐I , Mueller Robert F. , Griebe Thomas
Published: December 2004

An anaerobic upflow porous media biofilm reactor was designed to study the kinetics and stoichiometry of hydrogen sulfide production by the sulfate‐reducing bacterium (SRB) Desulfovibrio desulfuricans (ATCC 5575) as the first step for the modeling and control of formation souring (H2S) in oil field porous media. The reactor was a packed bed (50 × 5.5 cm) tubular reactor. Sea sand (140 to 375 μm) was used as the porous media. The initial indication of souring was the appearance of well‐separated black spots (precipitates of iron sulfide) in the sand bed. The blackened zones expanded radially and upward through the column. New spots also appeared and expanded into the cone shapes. Lactate (substrate) was depleted and hydrogen sulfide appeared in the effluent.

Analysis of the pseudo–steady state column shows that there were concentration gradients for lactate and hydrogen sulfide along the column. The results indicate that most of the lactate was consumed at the front part of the column. Measurements of SRB biomass on the solid phase (sand) and in the liquid phase indicate that the maximum concentration of SRB biomass resided at the front part of the column while the maximum in the liquid phase occurred further downstream. The stoichiometry regarding lactate consumption and hydrogen sulfide production observed in the porous media reactor was different from that in a chemostat. After analyzing the radial dispersion coefficient for the SRB in porous media and kinetics of microbial growth, it was deduced that transport phenomena dominate the souring process in our porous media reactor system. © 1994 John Wiley & Sons, Inc.
Novel protein a affinity matrix prepared from two‐dimensional protein crystals
Authors: Weiner Christian , Sára Margit , Sleytr Uwe B.
Published: December 2004

In this article, we describe a novel type of affinity matrix which was prepared by covalently binding Protein A to crystalline cell surface layers (S‐layers) from the gram‐positive Clostridium thermohydrosulfuricum L111‐69. S‐layers were used in the form of cell wall fragments, which were obtained by breaking whole cells by ultrasonification and removing the cell content and the plasma membrane. In these thimble shaped structures, revealing a size of 1 to 2 μm, the peptidoglycan‐containing layer was covered on both faces with a hexagonally ordered S‐layer lattice composed of identical glycoprotein subunits. After crosslinking the S‐layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein A. Quantitative determination confirmed that up to two Protein A molecules were bound per S‐layer subunit leading to a dense monomolecular coverage of the immobilization matrix with the ligand.

Affinity microparticles were capable of adsorbing lgG from solutions of purified preparations, from artificial lgG‐albumin mixtures, and from serum. The amount of lgG bound to affinity microparticles corresponded to the theoretical saturation capacity. Under appropriate conditions, up to 95% of the adsorbed lgG could be eluted again. Affinity microparticles were found to have an extremely low Protein A leakage and a high stability toward mechanical forces. Because pores in the S‐layer lattice revealed a size of 4 to 5 nm, immobilization of Protein A and adsorption of lgG was restricted to the outermost surface area. This excludes mass transfer problems usually encountered with affinity matrices prepared from amorphous polymers where more than 90% of the ligands are immobilized in the interior. © 1994 John Wiley & Sons, Inc.
Studies of on‐line viable yeast biomass with a capacitance biomass monitor
Authors: Austin Glen D. , Watson Rodney W. J. , D'Amore Tony
Published: December 2004

A commercially available biomass monitor has been employed in a number of applications. For capacitance monitors, a relationship between capacitance measurement and cell counts or colony forming units has been reported in the literature. However, for use as an online instrument, a more practical correlation with the biomass concentration is needed. In this study, we followed the batch growth of brewer's yeast and a correlation with viable biomass concentration (g DW/L) was demonstrated. This correlation was utilized with the capacitance biomass monitor in a control loop to maintain setpoint biomass levels in a cyclic reactor under perturbations. Not only did the system demonstrate the capability of the biomass monitor to control biomass in such a system, but it also confirmed the correlation reported in our earlier work. © 1994 John Wiley & Sons, Inc.
Partition coefficients of substrates and products and solvent selection for biocatalysis under nearly anhydrous conditions
Authors: Yang Zhen , Robb Donald A.
Published: December 2004

The effect of solvent on the activity of mushroom tyrosinase toward three substrates was studied at a constant water activity of either 0.74 or 0.86. No simple correlation was observed between enzyme activity and log P, but partition coefficients of substrate (Ps) and product (Pp) gave systematic relations with enzyme activity. When initial reaction rates were considered, there was a bellshaped relationship between enzyme activity and Ps with an optimal Ps for each substrate. This can be explained by assuming that the solvent affected the enzyme activity primarily by affecting the substrate concentration in the aqueous layer around the catalyst where the enzymic reaction occurs. When long‐term reaction rates were considered, a high Pp/Ps ratio was consistent with preservation of enzyme activity. © 1994 John Wiley & Sons, Inc.
Feeding heterogeneity in ciliate populations: Effects of culture age and nutritional state
Authors: Hatzis Christos , Srienc Friedrich , Fredrickson A. G.
Published: December 2004

We have used a novel approach in conjunction with flow cytometry to quantify the biological heterogeneity of populations of the ciliate Tetrahymena pyriformis. It was found that the rate of particle uptake of exponentially growing cells is not uniform among cells and partially correlated with cell size. The physiological state and growth history of the culture was found to affect to a large degree the population's feeding heterogeneity. Stationary phase populations exhibited more uniform feeding behavior, as cell aging affects all cells and effectively reduces their ability to feed. Cells that were removed from the growth medium and resuspended in nonnutritive medium exhibited a more heterogeneous feeding behavior. The starved cells were stimulated to feed at considerably higher rates, and the stimulatory effect was more pronounced for larger cells. It is therefore demonstrated that population heterogeneity has to be evaluated in conjunction with the populations growth state as it is determined by the history of the population's growth and nutritional state. © 1994 John Wiley & Sons, Inc.
Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique
Authors: Gama F. M. , Teixeira J. A. , Mota M.
Published: December 2004

An expeditious and accurate simplification of Stone and Scallan's solute exclusion technique was developed, thereby avoiding several sources of experimental error coupled with the determination of cellulose pore volume. Using this method, it is shown that cellulolytic enzymes do not enter into the micropores of five studied celluloses. These results suggestes that hydrolysis occurs initially at the external surface of the fibers. This surface area was calculated with the help of adsorption isotherms of bovine serum albumin. The obtained values for the different samples agree with the microscopically observed cellulose morphology. The correlation obtained by several authors relating cellulose porosity and its digestibility is explained as a consequence of the lower crystallinity and easier fragmentation of the more porous celluloses during hydrolysis. © 1994 John Wiley & Sons, Inc.
Ribosomal protein limitations in Escherichia coli under conditions of high translational activity
Authors: Laffend L. , Shuler M. L.
Published: December 2004

Details of the mechanism for ribosome synthesis have been incorporated in the single‐cell Escherichia coli model, which enable us to predict the amount of protein synthesizing machinery under different environmental conditions. The predictions agree quite well with available experimental data. The model predicts that ribosomal protein limitations are important when the translational apparatus is in high demand. Ribosomal RNA synthesis is induced by an increase in translational activity, which, in turn, stimulates ribosomal protein synthesis. However, as the demand increases still more, the ribosomal protein mRNA must compete with the plasmid mRNA for ribosomes, and the efficiency of translation of ribosomal proteins is reduced. © 1994 John Wiley & Sons, Inc.
Kinetic analysis of enzymatic chiral resolution by progress curve evaluation
Authors: Rakels J. L. L. , Romein B. , Straathof A. J. J. , Heijnen J. J.
Published: December 2004

The present study deals with kinetic modeling of enzyme‐catalyzed reactions by integral progress curve analysis, and shows how to apply this technique to kinetic resolution of enantiomers. It is shown that kinetic parameters for both enantiomers and the enantioselectivity of the enzyme may be obtained from the progress curve measurement of a racemate only.

A parameter estimation procedure has been established and it is shown that the covariance matrix of the obtained parameters is a useful statistical tool in the selection and verification of the model structure. Standard deviations calculated from this matrix have shown that progress curve analysis yields parameter values with high accuracies.

Potential sources of systematic errors in (multiple) progress curve analysis are addressed in this article. Amongst these, the following needed to be dealt with: (1) the true initial substrate concentrations were obtained from the final amount of product experimentally measured (mass balancing); (2) systematic errors in the initial enzyme concentration were corrected by incorporating this variable in the fitting procedure as an extra parameter per curve; and (3) enzyme inactivation is included in the model and a first‐order inactivation constant is determined.

Experimental verification was carried out by continuous monitoring of the hydrolysis of ethyl 2‐chloropropionate by carboxylesterase NP and the α‐chymotrypsin‐catalyzed hydrolysis of benzoylalanine mathyl ester in a pH‐stat system. Kinetic parameter values were obtained with high accuracies and model predictions were in good agreement with independent measurements of enantiomeric excess values or literature data. © 1994 John Wiley & Sons, Inc.
Glycosidase activities of the 293 and NS0 cell lines, and of an antibody‐producing hybridoma cell line
Authors: Gramer Michael J. , Goochee Charles F.
Published: December 2004

We have previously demonstrated that Chinese hamster ovary (CHO) cell lysates harbor sialidase, β‐galactosidase, β‐hexosaminidase, and fucosidase activities that can accumulate extracellularly in CHO cell culture, thereby potentially leading to extracellular modification of glycoprotein oligosaccharides. The sialidase activity in CHO cell lysates was surprisingly active and stable at pH 7.5, with a half‐life of 57 h at 37°C.

We have extended this work to determine whether 293, NS0, or hybridoma cell lysates contain similar glycosidase activities. The pH‐activity profiles of β‐galactosidase and β‐hexosaminidase in lysates of these three cell lines resemble the pH‐activity profiles for these enzymes in CHO cell lysate, whereas the pH‐activity profiles of sialidase and fucosidase appear to be cell‐type dependent. Sialidase activities were relatively stable at pH 4.5 in 293, NS0, and hybridoma cell lysates. However, the activities in 293 and NS0 cell lysates were unstable at pH 7.5, with no activity remaining after a 2‐h incubation at 37°C. The sialidase activity in hybridoma cell lysate was moderately stable at pH 7.5 with 30% of the activity remaining after a 2‐h incubation at 37°C. We conclude that the sialidase activites from 293, NS0, and hybridoma cells have characteristics similar to the vast majority of reported mammalian sialidase activities, and that these activities are markedly differant from the CHO cell sialidase activity.

Finally, sialidase, β‐galactosidase, β‐hexosaminidase, and fucosidase activities were measured at pH 7 in cell‐free bioreactor supernatants of the hybridoma cell line. As previously observed in CHO cell culture, all four glycosidase activities were present in the hybridoma supernatants. However, the sialidase activity in hybridoma supernatant was an order of magnitude lower than in CHO cell culture supernatant despite the fact that the hybridoma cell lysis rate was an order of magnitude higher. © 1994 John Wiley & Sons, Inc.
Extraction of cephalosporin C from whole broth and separation of desacetyl cephalosporin C by aqueous two‐phase partition
Authors: Yang Wu‐Yung , Lin Chun‐Der , Chu I‐Ming , Lee Chau‐Jen
Published: December 2004

Cephalosporin C was extracted from diluted or whole broth by PEG/salt aqueous two‐phase systems. Parameters such as PEG molecular weight, salt type, pH, and salt concentration were investigated for finding a suitable extraction system. In PEG 600/ammonium sulfate or phosphate systems, Kc (partition coefficienct of cephalosporin C) was observed to be larger than 1, with Kd (partition coefficient of desacetyl cephalosporin C) being smaller than 1. The particular values of these coefficients would imply that the difficult separation of cephalosporin C and desacetyl cephalosporin C could possibly be achieved via the aqueous two‐phase extraction. The addition of surfactants, water‐miscible solvents, and neutral salts for enhancement of the separation efficiency was also investigated. The addition of surfactants to the system did not affect the separation efficiency substantially. Kc would increase whereas Kd decreased as a result of the addition of acetone, MeOH, EtOH, IPA, and n‐BuOH. Meanwhile both Kc and Kd would decrease whenever neutral salts, NaCl, KCl, Kl, or KSCN, were added. The partitioning behavior of cephalosporin C and desacetyl cephalosporin C in filtered, whole, and different batches of broth was notably quite similar to that of diluted broth. The recovery yield of cephalosporin C in whole broth extraction was observed to be a function of centrifugal force used in phase separation. © 1994 John Wiley & Sons, Inc.
Whole skeletal muscle transplantation: Mechanisms responsible for functional deficits
Authors: Faulkner John A. , Carlson Bruce M. , Kadhiresan Veerichetty A.
Published: December 2004

One aspect of tissue engineering of skeletal muscle involves the transposition and transplantation of whole muscles to treat muscles damaged by injury or disease. The transposition of whole muscles has been used for many decades, but since 1970, the development of techniques for microneurovascular repair has allowed the transplantation of muscles invariably result in structural and functional deficits. The deficits are of the greatest magnitude during the first month, and then a gradual recovery results in the stabilization of structural and functional variables between 90 and 120 days. In stabilized vascularized grafts ranging from 1 to 3 g in rats to 90 g in dogs, the major deficits are ∼25% decrease in muscle mass and in most grafts ∼40% decrease in maximum force. The decrease in power is more complex because it depends on both the average shortening force and the velocity of shortening. As a consequence, the deficit in maximum power may be either greater or less than the deficit in maximum force. Tenotomy and repair are the major factors responsible for the deficits.

Although the data are limited, skeletal muscle grafts appear to respond to training stimuli in a manner no different from that of control muscles. The training stimuli include traditional methods of endurance and strength training, as well as chronic electrical stimulation. Transposed and transplanted muscles develop sufficient force and power to function effectively to: maintain posture; move limbs; sustain the patency of sphincters; partially restore symmetry in the face; or serve as, or drive, assist devices in parallel or in series with the heart.
Effects of substratum morphology on cell physiology
Authors: Singhvi Rahul , Stephanopoulos Gregory , Wang Daniel I. C.
Published: December 2004

Among the host of substratum properties that affect animal cell behavior, surface morphology has received relatively little attention. The earliest effect of surface morphology on animal cells was discovered almost a century ago when it was found that cells became oriented in response to the underlying topography. This phenomenon is now commonly known as contact guidance. From then until very recentrly, little progress has been made in understanding the role of surface morphology on cell behavior, primarily due to a lack of defined surfaces with uniform morphologies. This problem has been solved recently with the development of photolithographic techniques to prepare substrata with well defined and uniform surface morphologies. Availability of such surfaces has facilitated systematic in vitro experiments to study influence of surface morphology on diverse cell physiological aspects such as adhesion, growth, and function. For example, these studies have shown that surfaces with uniform multipls parallel grooves can enhance cell adhesion by confining cells in grooves and by mechanically interlocking them. Several independent studies have demosterated that cell shape is a major determinant of cell growth and function. Because surface morphology has been shown to modulate the extent of cell spreading and cell shape, its effects on cell growth and function appear to be mediated via this biological coupling between cell shape and function. New evidence in the cell biology literature is emerging to suggest that surface morphology could affect other cell behavioral properties such as post‐translational modifications. Further elucidation of such effects will enable better designs for implant and cell culture substrata.
N‐acetylglucosamine and adenosine derivatized surfaces for cell culture: 3T3 fibroblast and chicken hepatocyte response
Authors: Gutsche A. T. , Parsons‐Wingerter P. , Chand D. , Saltzman W. M. , Saltzman W. M. , Leong K. W.
Published: December 2004

3T3 fibroblasts and primary chicken hepatocytes were cultured on derivatized polystyrene surfaces to examine the effect of cell‐specific ligands on cellular morphology and growth. Surfaces were prepared by derivatizing chloromethylated polystyrene with N‐acetylglucosamine (GlcNAc; recognized by the chicken asialoglycoprotein receptor) and adenosine (not recognized by adult hepatocytes). These surfaces were compared with tissue culture polystyrene (TCPS), acid‐cleaned glass, and the unmodified chloromethylated polystyrene. The spreading, cytoskeletal structure and growth of the fibroblasts following attachment to these surfaces were examined. The extent of attachment, total protein levels, and DNA contents for surfaces‐attached chicken hepatocytes were also measured. Fibroblast spreading was greatest on polymer surfaces derivatized with GlcNAc, whereas cytoskeletal structure and growth rate were independent of surface chemistry. Although chicken hepatocytes attached most efficiently to the GlcNAc derivatized polymer, the total protein and DNA levels of the surface‐attached cells were not affected. In anticipation of the application of these polymers for cell culture and hybrid artificial organ design, the GlcNAc‐derivatized polystryrene was fabricated into porous microcarriers. Fibroblasts grew avidly on the microcarriers, whereas chicken hepactocytes adhered well to the formed large aggregates arounds the microcarriers.
Filtration‐based perfusion of hybridoma cultures in protein‐free medium: Reduction of membrane fouling by medium supplementation with DNase I
Authors: Mercille Sylvain , Johnson Mark , Lemieux Réal , Massie Bernard
Published: December 2004

In this study, a filtration‐based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma cells grown in protein‐free medium. It was found that the use of protein‐free medium for perfusion culture generated the formation of numerous visible suspended particles consisting of dead cells and cellular debris aggregated into fibrous material. Surprisingly high apparent viabilities were observed in such protein‐free cultures. In addition, membrane fouling occurred more rapidly in protein‐free medium than in conventional serum‐supplemented medium. By the addition of deoxyribonuclease I (DNase I) to the protein‐free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total cell population more accurately. Moreover, DNase I significantly reduced the fouling of filtration membranes, and that, for two different types of separation systems (cross‐flow and vortex‐flow filtration) and two different types of membranes (polycarbonate and hydrophilized polysultone). From these results, it is clear that the presence of DNA fragments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex‐flow filtration module than with a static plate‐and‐frame cross‐flow filtration module. The use of vortex‐flow filtration of conjuction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or antibody retention and with a constant permeate IgM concentration of 250 mg/L. Hybridomacells appeared to gradually adapt to increasing rotational speed in the vortex‐flow filtration module.
A metabolic model of cellular energetics and carbon flux during aerobic Escherichia coli fermentation
Authors: Ko Yun‐Fei , Bentley William E. , Weigand William A.
Published: December 2004

An integrated metabolic model for the production of acetate by Escherichia coli growing on glucose under aerobic conditions was presented previously (Ko et al., 1993). The resulting model equations can be used to explain phenomena often observed with industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low dissolved oxygen concentration, a high specific growth rate, or a combination of these conditions. However, several questions still need to be addressed. First, cell composition is growth rate and media dependent. Second, the macromolecular composition varied between E. coli strains. And finally, a model that represents the carbon fluxes between the Embden–Meyerhof–Parnas (EMP) and the hexose monophosphate (HMP) pathways when cells are subject to internal and/or external stresses is still not well defined. In the present work, we have made an effort to account for these effects, and the resulting model equations show good agreement for wild‐type and recombinant E. coli experimental data for the acetate concentration, the onset of acetate secretion, and cell yield based on glucose. These results are useful for optimizing aerobic E. coli fermentation processes. More specifically, we have determined the EMP pathway carbon flux profiles required by the integrated metabolic model for an accurate fit of the acetic acid profile data from a wild‐type E. coli strain ML308. These EMP carbon flux profiles were correlated with a dimensionless measurement of biomass and then used to predict the acetic acid profiles for E. coli strain F‐122 expressing human immunodeficiency virus‐(HIV528) β‐galactosidase fusion protein. The effect of different macromolecular compositions and growth rates between these two E. coli strains required a constant scaling factor for improved quantitative predictions.
Mammalian cell damage in a novel membrane bioreactor
Authors: Millward H. R. , Bellhouse B. J. , Nicholson A. M. , Beeton S. , Jenkins N. , Knowles C. J.
Published: December 2004

The experimental study has assessed a novel membrane bioreactor for mammalian cell culture. In the absence of a gas phase, the key features of cell damage associated with laminar and turbulent flow have been identified. The bioreactor employs a dimpled membrane in order to enhance transverse mixing in a narrow channel, but a fall in viable cell density has been observed at Reynolds numbers above Re = 83. In the laminar flow regime wall shear is the critical mechanism and an accurate calculation of shear rate in a complex channel has been achieved using the Reynolds analogy. Flow generating a wall shear rate in excess of 3000 s−1 has been shown to cause damage. Power dissipation measurements have been used to distinguish between laminar and turbulent flow and also to predict Kolmogorov eddy lengths. An additional turbulent bulk stress damage mechanism at higher Reynolds numbers (Re > 250) results in a very rapid fall in viable cell density.
Enzymatic catalysis in heterogenous mixtures of substrates: The role of the liquid phase and the effects of “Adjuvants”
Authors: López‐Fandiño Rosina , Gill Iqbal , Vulfson Evgeny N.
Published: December 2004

The physicochemical mechanism of protease‐catalyzed peptide synthesis in heterogenous etuectic mixtures of substrates has been examined by a combination of microscopic techniques. Using a number of model reactions of dipeptide amide synthesis, it was determined that liquid phase catalysis was mostly, if not exclusively, responsible for the observed conversion of substrates. Furthermore, the formation of liquid or semiliquid eutectics was an important requirement for the occurrence of those reactions where both substrates were solids in the pure state. The addition of small quantities of hydrophilic solvents (adjuvants) often resulted in significat improvements in the rates and yields of the reactions. This was due to the ability of these adjuvants to promote the formation of eutectics, thereby increasing the proportion, as well as affecting the composition the properties, as well as affecting the composition and properties of the liquid phase. © 1994 John Wiley & Sons, Inc.
Cybernetic model for synthesis of poly‐β‐hydroxybutyric acid in Alcaligenes eutrophus
Authors: yoo Seung , Kim Woo‐Sik
Published: December 2004

The pathway of poly‐β‐hydroxybutyric acid (PHB) exhibits a mode of transcriptional control induced by environmental stress. A new cybernetic model for coordinated regulation of stress‐induced metabolism was developed to predict the growth and the synthesis of PHB in Alcaligenes eutrophus. A plausible objective for this control is optimization of acetyl‐CoA utilization so that the cells have a high degree of flexibility in their catabolism. The state equation for key protein synthesis was assumed to have a dependence on the nonlinear control variable. The proposed model can demonstrate the mixed‐growth‐associated synythesis of PHB. Reported unstructured models were compared statistically with the result of the simulation derived from the proposed model using the experimental data of this study and the literature. The proposed model appeared to provide an excellent description for the overall fermentation range. © 1994 John Wiley & Sons, Inc.
The effects of pH and ionic strength on the partitioning of four proteins in reverse micelle systems
Authors: Andrews B. A. , Pyle D. L. , Asenjo J. A.
Published: December 2004

Four proteins with different physicochemical properties have been partitioned in reversed micelle systems: thaumatin, ribonuclease A, soybean trypsin inhibitor, and α‐lactalbumin. The organic phase was formed by sodium salt (AOT) in isooctane, and the aqueous phase contained KCl, KBr, MgCl2, or NaCl. Aqueous phase pH was varied between 2 and 13 and ionic strength from 0.1 to 1.0 M. Small changes in pH [around the isoelecric point (pl)] were found to influence the solubilization of ribonuclease A and trypsin inhibitor, but for thaumatin the pH change necessary to affect partition was much greater as a consequence of the difference in net charge (titration curves) of these protein molecules as pH changes. The type of ions present in the system was also a determining factor for partition; the larger ions (K+) produced more electrostatic screening and hence less protein solubilization than the smaller ions (Na+). With changes in ionic strength surface hydrophobicity was a dominant factor affecting solubilization of thaumatin in NaCl‐containing systems at high pH. Charge distribution and hydrophobicity are thought to be important parameters when partitioning the protein α‐lactalbumin. © 1994 John Wiley & Sons, Inc.
Stabilizing effect of amphiphilic excipients on the freeze‐thawing and freeze‐drying of lactate dehydrogenase
Authors: Izutsu Ken‐ichi , Yoshioka Sumie , Terao Tadao
Published: December 2004

The effects of amphiphilic excipients on the inactivation of lactate dehydrogenase (LDH) during freeze‐thawing and freeze‐drying were studied. Some amphiphilic excipients such as hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD), CHAPS, polyethylene glycol (PEG) 3350, and sucrose fatty acid monoester prevented LDH inactivation during freeze‐thawing and freeze‐drying at a lower concentration than sugars and amino acids. Polyoxyethylene 9 lauryl ether and PEG 400 protected LDH during freeze‐thawing but not during freeze‐drying. The buffer concentration of the solution to be freeze‐dried (10, 50, and 200 mM) affected the stabilizing effect of trehalose, but not that of HP‐β‐CD. © 1994 John Wiley & Sons, Inc.
Anaerobic acidogenesis of primary sludge: The role of solids retention time
Authors: Elefsiniotis Panagiotis , Oldham William K.
Published: December 2004

This research investigates the effect of solids retention time (SRT) on the acid‐phase anaerobic digestion of primary sludge. A series of experiments were conducted using two continuous‐flow 3‐L units with the following configuration: a completely mixed reactor (CMR) with clarifier and solids recycle and an upflow anaerobic sludge blanket (UASB) reactor. Results show that C2 to C5 volatile fatty acids (VFA) were the predominant compounds formed. At a constant hydraulic retention time (HRT) of 12 h, variation in SRT from 10 to 20 days resulted in a slight increase in VFA production in both systems, but at a shorter SRT (5 days) a drastic drop in acid production was observed. In addition, the percent distribution of VFA was to some extent affected by the change in SRT. On the other hand, organic matter degradation [measured by the chemical oxygen demand (COD) specific solubilization rate or the percent volatile suspended solids (VSS) reduction] appeared to be independent of SRT, at least in the range investigated. The percent soluble COD in the form of VFA, however, increased steadily with increasing SRT, approaching the 90% level at 20 days. The remaining soluble COD in the effluent from these systems may be mainly attributed to metabolic intermediates and unused soluble substrate. © 1994 John Wiley & Sons, Inc.
Two‐phase airlift fermentor operation with elicitation for the enhanced production of enzophenanthridine alkaloids in cell suspensions of Escherichia californica
Authors: Byun Sang Yo , Pedersen Henrik
Published: December 2004

Approaches to increasing the productivity of benzophenanthridine alkaloids in suspension cultures in Escherichia californica were made in an airlift fermentor under different culture conditions. Elicitation with yeast extract elicitor reduced the time required to obtain a certain amount of alkaloid production. In a two‐phase airlift fermentor with compounded silicone fluid, total alkaloid concentration in silicone fluid was 153.1 mg/L and that in the aqueous cellular phase was 8.2 mg/L at day 21 from inoculation. The large accumulation capacity of silicone fluid made it possible to store correspondingly large amounts of total alkaloid and increased the alkaloid production. Act day 21 from inoculation, the volumetric alkaloid productivity and the netproduction in a two‐phase airlift fermentor were 1.4 and 1.5 times higher than those of normal airlift fermentor operation. This performance was furthermore enhanced by elicitation. Elicitation in two‐phase airlift fermentor operation increased the volumetric productivity and the new production 3.3‐ and 3.5‐fold compared to those of normal airlift fermentor operation. © 1994 John Wiley & Sons, Inc.
Physicochemical properties of the matrix proteins of three main culture vehicles
Authors: Andrews A. T. , Noble I. , Keeratatipibul S. , Asenjo J. A.
Published: December 2004

The protein components of three industrial recombinant expression systems: Escherichia coli, Saccharomyces cerevisiae, and a mammalian cell culture supernatant of CHO cells were characterized in terms of their molecular weight, isoelectric point, and relative surface hydrophobicity. Identification of individual proteins was done by reference to their position in protein band profiles by polyacrylamide gel electrophoresis (PAGE) of the crude material. This permitted a rapid and facile assignment of quantitative values for these three parameters to all the major protein components in these materials. Because it is the indigenous proteins in expression systems that will form the bulk of any impurities in the product, once the values of these parameters are known for any target recombinant protein, the data obtained will enable appropriate expression systems to be chosen for minimizing amounts of potential contaminants and reducing downstream processing requirements and costs. The data will also indicate which fractionation steps (i.e., charge, size or hydrophobicity‐based) are likely to be best for distinguishing between target and contaminant proteins, thus aiding and early removal of the maximum quantities of undesired protein to bring subsequent bioseparation steps down in scale and cost and up in terms of efficiency. © 1994 John Wiley & Sons, Inc.
Modeling approach to control of carbohydrate metabolism during citric acid accumulation by Aspergillus niger: I. Model definition and stability of the steady state
Authors: Torres Néstor V.
Published: December 2004

A model of the carbohydrate metabolism and the anaplerotic synthesis of oxalacetate in Aspergillus niger, under conditions of citric and accumulation, is presented. In this first article we set the stage for subsequent analysis within the framework of the biochemical system theory (BST): we formulate the model and develop the system representation in power law forms, showing that the steady state is locally stable. In the second article, the control structure of the system is described and a rationale for the optimization of the process is developed. © 1994 John Wiley & Sons, Inc.
Interactions of microbial biofilms with toxic trace metals: 2. Prediction and verification of an integrated computer model of lead (II) distribution in the presence of microbial activity
Authors: Hsieh Ke Ming , Murgel George A. , Lion Leonard W. , Shuler Michael L.
Published: December 2004

The interfacial interactions of a toxic trace metal, Pb, with a surface modified by a marine film‐forming bacterium, Psedomonas atlantica, were predicted by a structured biofilm model used in conjunction with a chemical speciation model. The validity of the integrated model was tested for batch and continuous operations. Dynamic responses of the biophase due to transient lead concentration increases were also stimulated. The reasonable pre dictions achieved by the model demonstrate its utility in describing trace metal distributions in complex systems where the adsorption properties of inorganic surfaces are modified by adherent bacteria production of extracellular polymers. © 1994 John Wiley & Sons, Inc.
Optimization of microbial poly(3‐hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform
Authors: Hahn Sei Kwang , Chang Yong Keun , Kim Beom Soo , Chang Ho Nam
Published: December 2004

Optimization was carried out for the recovery of microbiol poly(3‐hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90‐min treatment time, and a 1:1 (v/v) chloroform‐to‐aqueous‐phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, Mn of recovered PHB was about 300,000 and the weight average molecular weight Mw was about 1,020,000, compared to the original Mn of 530,000 and Mw of 1,272,000. The moderate decrease in both Mn and Mw might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in Mw probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. © 1994 John Wiley & Sons, Inc.
Hydrolysis of triglyceride by the whole cell ofPseudomonas putida3SK in two‐phase batch and continuous reactors systems
Authors: Lee Seoung Yong , Rhee Joon Shick
Published: December 2004

Batch and continuous hydrolysis of olive oil in an organic‐aqueous two‐phase system using the live whole cell ofPseudomonas putida3SK as a source of a lipase is investigated. The strain was not only fully viable and grown well, but also produced extracellular lipase simultaneously. The degree of hydrolysis, depending on olive oil concentration in the solvents, was maximal at 13.5% (w/v) and decreased with the increase of the substrate concentration. At the optimal condition, a degree of hydrolysis higher than 95% was achieved with 24 h at 30°C when the reaction was carried out in a two‐phase batch stirred reactor. For long‐term operation a continuous stirred reactor was designed. When the reaction was carried out in a continuous stirred reactor, the degree was hydrolysis reached 86% at a dilution rate of 0.2 h−1. Satisfactory performance of a two‐phase bioreactor was obtained in a long‐term continous operation, which lasted for at least 30 days by feeding organic solvent containing olive oil and aqueous media separately. © 1994 John Wiley & Sons, Inc.
Partitioning of pristinamycins in aqueous two‐phase systems: A first step toward the development of antibiotic production by extractive fermentation
Authors: Paquet V. , Myint M. , Roque C. , Soucaille P.
Published: December 2004

The partitioning of pristinamycins was studied in dextran and polyethylene glycol (PEG) aqueous two‐phases systems. Pristinamycins partitioned preferentially into the PEG‐rich top phase. The partition coefficient was independent of molar mass of PEG and dextran and of antibiotic concentration, but, increased exponentially with the tieline length of the system. Partition of pristinamycins was greatly improved when fatty acids esters of PEG were mixed with PEG. In such mixtures, the partition of coefficient increased up to a value of 24, dependent on the carbon chain length of fatty acids and the modified PEG concentrations. Moreover, in such system, the two groups of pristinamycins, I and II, were extracted in accordance with their hydrophobicity. Recovery of pristinanamycins produced by Streptomyces pritinaespiralis in a fermentation broth was achieved with a dextran/PEG system. Cells were confined into the bottom phase and pristinamycins partitioned in the top phase. However, due to binding of the pristinamycins to the cells, the partition coefficient was slightly lower than of pure antibiotics solutions. © 1994 John Wiley & Sons, Inc.
Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams
Authors: Wongsamuth Raviwan , Doran Pauline M.
Published: December 2004

Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell‐free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam‐C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced kLa values in bubble‐column and stirred‐tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head‐space volume and tolerable foam levels must be considered to optimize biomass production. © 1994 John Wiley & Sons, Inc.
Transport of bacteria in porous media: II. A model for convective Transport and growth
Authors: Sarkar A. K. , Georgiou George , Sharma Mukul M.
Published: December 2004

A model is presented for the coupled processes of bacterial growth and convective transport of bacteria has been modeled using a fractional flow approach. The various mechanisms of bacteria retention can be incorporated into the model through selection of an appropriate shape of the fractional flow curve. Permeability reduction due to pore plugging by bacteria was simulated using the effective medium theory. In porous media, the rates of transport and growth of bacteria, the generation of metabolic products, and the consumption of nutrients are strongly coupled processes. Consequently, the set of governing conservation equations form a set of coupled, nonlinear partial differential equations that were solved numerically. Reasonably good agreement between the model and experimental data has been obtained indicating that the physical processes incorporated in the model are adequate. The model has been used to predict the in situ transport and growth of bacteria, nutrient consumption, and metabolite production. It can be particularly useful in simulating laboratory experiments and in scaling microbial‐enhanced oil recovery or bioremediation processes to the field. © 1994 John Wiley & Sons, Inc.
Enzymatic hydrolysis of whey proteins: I. Kinetic models
Authors: Gonzàlez‐Tello P. , Camacho F. , Jurado E. , Páez M. P. , Guadix E. M.
Published: December 2004

We have studied the enzymatic hydrolysis of whey proteins at pH 8 and50°C with two proteases of bacterial origin, MKC Protease 660 L, and one of animal origin, PEM 2500 S. Our results show that a greater degree of hydrolysis is achieved under the same experimental conditions with the bacterial proteases than with the animal one. In our interpretation of the results we propose a mechanism in which the hydrolytic reaction is a zero‐order one for the substrate, and the enzyme denaturalizes simultaneously via a second‐order kinetic process due to free enzyme attacking enzyme bound to the substrate. Our results also indicate that there is an irreversible serine‐protease inhibitor in whey proteins. © 1994 John Wiley & Sons, Inc.
Influence of strain and medium composition on filtration of escherichia coli suspensions
Authors: Junker B. H. , Timberlake S. , Bailey F. J. , Reddy J. , Prud'homme R. , Gbewonyo K.
Published: December 2004

Cross‐flow filtration of Escherichia coli strains was examined at the laboratory and pilot scales using Romicon 500,000 molecular‐weight‐cutoff hollow fiber membranes. Both the series resistance and macrosolute polarization models were employed to compare performances. Total dissolved solids content above 90 g/L and viscosity above 1.1 × 10−3 paċ s of cell‐free culture media were found to decrease average filtration fluxes by over 60% both in the absence and presence of cells. Broth filtration with culture media of dissolved solids levels below 80 g/L were influenced to a greater extent by harvest cell density. The collodial nature of the complex nutrient responsible for the total solids increase affected prediction of filtration performance. Differences in strain filterability were observed with JM109 preferred over DH5 in high solids‐containing media and RR1 preferred over JM109 in low dissolved solids‐containing media. Their research demonstrates the importance of cell strain and media selection in the performance of early downstream processing steps. © 1994 John Wiley & Sons, Inc.