bluuebunny/crossref_metadata_2025 huggingface dataset:
High cell density cultivation of Pseudomonas oleovorans: Growth and production of poly (3‐hydroxyalkanoates) in two‐liquid phase batch and fed‐batch systems
Authors: Preusting Hans , van Houten Renze , Hoefs André , van Langenberghe Eddy Kool , Favre‐Bulle Olivier , Witholt Bernard
Published: December 2004

Pseudomonas oleovorans is able to accumulate poly(3‐hydroxyalkanoates) (PHAs) under conditions of excess n‐alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed‐batch cultures.

To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n‐octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g ·L−1 dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30°−18°C, cell growth continued to a final cell density of 35 g · L−1 due to a lower oxygen demand of the cells at this lower incubation temperature.

To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (kLa) was determined during growth of P. oleovorans on n‐octane. Since the stirrer speed and airflow were increased during growth of the organism, the kLa also increased, reaching a constant value of 0.49 s−1 at maximum airflow and stirrer speed of 2 L · min−1 and 2500 rpm, respectively. This kLa value suggests that oxygen transfer is very efficient in our stirred tank reactor.

Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed‐batch cultures was studied. The cells were first grown batchwise to a density of 6 g · L−1, after which a nutrient feed, consisting of (NH4)2SO4 and MgSO4, was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH4+ per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g · L−1 was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g · L−1 and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. © 1993 John Wiley & Sons, Inc.
Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction
Authors: Vallino Joseph J. , Stephanopoulos Gregory
Published: December 2004

The two main contributions of this are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of bansal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a through literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis‐directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support fluxes during growth and lysine overproduction branch point does not appear to limit lysine synthesis. © 1993 John Wiley & Sons, Inc.
Phenol removal from waste gases with a biological filter by Pseudomonas putida
Authors: Zilli M. , Converti A. , Lodi A. , Borghi M. Del , Ferraiolo G.
Published: December 2004

The purpose of this study is to investigate the feasibility of biologically removing phenol from waste gases by means of a biofilter using a Pseudomonas putida strain. Two series of both batch and continuous tests have been performed in order to ascertain the microbial degradation of phenol. For the preliminary batch tests, carried out in order to test the effective feasibility of the process and to investigate their kinetic behavior, two different microbial cultures belonging to the Pseudomonas genus have been employed, a heterogeneous culture and a pure strain of P. putida. The results of these comparative investigation showed that the pure culture is more efficient than the mixed one, even when the latter has undergone three successive acclimatization tests. The continuous experiments have been conducted during a period of about 1 year in a laboratory‐scale column, packed with a mixture of peat and glass beads, and utilizing the pure culture of P. putida as microflora and varying the inlet phenol concentration from 50 up to 2000 mg m−3. The results obtained show that high degrees of conversion can be obtained (0.93/0.996) operating at a residence time of 54 s. © 1993 John Wiley & Sons, Inc.
Cadmium biosorption by Saccharomyces cerevisiae
Authors: Volesky B. , May H. , Holan Z. R.
Published: December 2004

Cadmium uptake by nonliving and resting cells of Saccharomyces cerevisiae obtained from aerobic or anaerobic cultures from pure cadmium‐bearing solutions was examined. The highest cadmium uptake exceeding 70 mg Cd/g was observed with aerobic baker's yeast biomass from the exponential growth phase. Nearly linear sorption isotherms featured by higher sorbing resting cells together with metal deposits localized exclusively in vacuoles indicate the possibility of a different metal‐sequestering mechanism when compared to dry nonliving yeasts which did not usually accumulate more than 20 mg Cd/g. The uptake of cadmium was relatively fast, 75% of the sorption completed in less than 5 min. © 1993 Wiley & Sons, Inc.
Factor affecting enzyme characteristics of bilirubin oxidase suspensions in organic solvents
Authors: Skrika‐Alexopoulos Eleftheria , Freedman Robert B.
Published: December 2004

The activity of bilirubin oxidase toward bilirubin was studied in a liquid/solid two‐phase low‐water organic system using a simple spectrophotometric assay to follow the reaction. The enzyme was lyophilized from aqueous solution before being suspended in the organic solvent reaction medium. The activity was significantly influenced by the properties of the aqueous medium from which the enzyme was lyophilized, specifically its pH, and the quantity and nature of the buffering species. Analyses of these effect showed that the role of buffering species in such systems went beyond their effect in fixing the protonation state of the enzyme. The activity was also influenced by the quantity of water added to the organic solvent reaction medium. The reaction was shown to follow Michaelis‐Menten Kinetics, andKmandkcatwere determined. The liquid/solid two‐phase system studied was extensively compared to a previously studied water‐in‐oil microemulsion system © 1993 Wiley & Sons, Inc.
Pulsatile flow filtration of yeast cell debris: Influence of preincubation on performance
Authors: Wu D. , Howell J. A. , Field R. W.
Published: December 2004

The recovery of proteins from yeast cell debris suspension was investigated using a vortex mixing technique based on the combination of oscillatory flow and a baffled flat‐sheet microfiltration system. For this system, increased protein transmission was obtained through the use of low transmembrane pressures and the preincubation of the yeast cell debris feed suspension at 30°C. Furthermore, a plateau in the flux‐time curve was observed. In the absence of baffles and pulsations, preincubation had a little effect. © 1993 Wiley & Sons, Inc.
Influence of mold growth on the pressure drop in aerated solid state fermentors
Authors: Auria Richard , Morales Marcia , Villegas Elba , Revah Sergio
Published: December 2004

The measurement of pressure drop(ΔP) across an aerated fermentation bed is proposed as alternative on‐line sensor for the qualitative and, in some cases, quantitative, macroscopic changes in a static solid state fermentor. An increase in the ΔP is correlated with the evolution of the different phases of Aspergillus niger growth: germination, vegetative growth, limitation, and sporulation, we observed in the microscope. For the case where the support is not modified during the fermentation and the water content remains constant, i.e., a synthetic resin (Amberlite IRA‐900), the gas phase permeability of the bed is directly related to the biomass content. For example, the permeability of the bed is reduced to 5% of the initial value when biomass attains 21 mg dry biomass/g dry support. Biomass was appropriately predicted from the ΔP measurements in an independent test. Experiments with different initial sucrose solution concentrations showed that biomass could not be produced beyond a certain level (21.5 mg dry biomass/g dry support) which suggests steric limitations. For the case of wheat bran and cane bagasse, the increase in ΔP was related qualitatively to the evolution in the growth and the morphology of the mold . © 1993 Wiley & Sons, Inc.
The kinetics of cometabolism
Authors: Criddle Craig S.
Published: December 2004

Experimental observations indicate that the rates of cometabolic transformation are linked to the consumption of growth substrate during growth and to the consumption of cell mass and/or energy substrate in the absence of growth substrate. Three previously proposed models (models 1 through 3) describing the kinetics of cometabolism by resting cells are compared, and the interrelationships and underlying assumptions for these models are explored. Models 1 to 3 are shown to converge at high concentrations of the nongrowth substrate. An expression describing nongrowth substrate transformation in the presence of growth substrate is proposed, and this expression is integrated with an expression for cell growth to give a single unstructured model (model 4) that encompasses models 1 to 3 and describes cometabolism by both resting and growing cells. Model 4 couples transformation of nongrowth substrate to consumption of growth substrate and biomass, and predicts that cometabolism will result, and decreased specific growth rates for a cometabolizing population. Competitive inhibition can also be incorporated in the model. Experimental aspects of model calibration and verification are discussed. The need for models that distinguish between the exhaustion of cell activity and cell death is emphasized. © 1993 Wiley & Sons, Inc.
A mathematical model for dynamic simulation of anaerobic digestion of complex substrates: Focusing on ammonia inhibition
Authors: Angelidaki I. , Ellegaard L. , Ahring B. K.
Published: December 2004

A mathematical model for anaerobic degradation of complex organic material, such as manure, has been developed. The model includes an enzymatic hydrolytic step and four bacterial steps and involves 12 chemical compounds. The model focuses on ammonia inhibition and includes a detailed description of pH and temperature characteristics in order to accurately simulate free ammonia concentration. Free ammonia and acetate constitute the primary modulating factors in the model. The model has been applied for the simulation of digestion of cattle manure in continuously stirred tank reactors (CSTRs), and results compare favorably with experimental data. © 1993 John Wiley & Sons, Inc.
Measurements of conformational changes during adhesion of lipid and protein (polylysine and S‐layer) surfaces
Authors: Leckband Deborah , Chen You‐Lung , Israelachvili Jacob , Wickman H. Hollis , Fletcher Madilyn , Zimmerman Robert
Published: December 2004

The adhesion forces between various surfaces were measured using the “surface forces apparatus” technique. This technique allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. Three types of biological surfaces were prepared by depositing various lipid‐protein monolayers (with thicknesses ranging from 1 to 4 nm) on the inert, molecularly smooth mica surface: (i) hydrophobic lipid monolayers; (ii) amphiphilic polyelectrolyte surfaces of adsorbed polylysine; and (iii) deposited bacterial S‐layer proteins. The adhesion, swelling, and wetting properties of these surfaces was measured as a function of relative humidity and time. Initial adhesion is due mainly to the van der Waals forces arising from nonpolar (hydrophobic) contacts. Following adhesive contact, significant molecular rearrangements can occur which alter their hydrophobic–hydrophilic balance and increase their adhesion with time. Increased adhesion is generally enhanced by (i) increased relative humidity (or degree of hydration); (ii) increased contact time; and (iii) increased rates of separation. The results are likely to be applicable to the adhesion of many other biosurfaces, and show that the hydrophobicity of a lipid or protein surface is not an intrinsic property of that surface but depends on its environment (e.g., on whether it is in aqueous solution or exposed to the atmosphere), and on the relative humidity of the atmosphere. It also depends on whether the surface is in adhesive contact with another surface and—when considering dynamic (nonequilibrium) conditions—on the time and previous history of its interaction with that surface. © 1993 John Wiley & Sons, Inc.
Observations of aerobic, growing escherichia coli metabolism using an on‐line nuclear magnetic resonance spectroscopy system
Authors: Chen Ruizhen , Bailey James E.
Published: December 2004

An experimental system has been constructed which enables on‐line measurements of phosphorus‐31 (31P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high‐cell‐density cultures. 31P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane ΔpH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. © 1993 John Wiley & Sons, Inc.
Analytical effectiveness calculations concerning the degradation of an inhibitive substrate by a steady‐state biofilm
Authors: van Ede C. J. , Bollen A. M. , Beenackers A. A. C. M.
Published: December 2004

A reaction engineering model for the degradation of an inhibitory substrate by a steady‐state biofilm is presented. The model describes both the metabolic rate controlling behavior of this substrate in the biofilm and the effect of diffusion limitation caused by an arbitrary substrate on the active biofilm thickness. An analytical expression for the biocatalyst effectiveness factor is presented on the basis of Pirt kinetics for cell maintenance, first order substrate inhibition kinetics, and zero order substrate consumption kinetics. The proposed expression for the biocatalyst effectiveness factor is much more convenient to incorporate into a macroreactor model than the numerical alternatives. Simple criteria are presented to check the applicability of the model in case of true Monod kinetics. The analytical solution is expected to be particularly applicable to processes where a low soluble organic substrate controls the biomass growth, a situation which is often met in wastewater purification processes of industrial importance. The degradation of phenol by Pseudomonas sp. is treated as an example. © 1993 John Wiley & Sons, Inc.
A model system for a fluorometric biosensor using permeabilized Zymomonas mobilis or enzymes with protein confined dinucleotides
Authors: Thordsen O. , Lee S. J. , Degelau A. , Scheper T. , Loos H. , Rehr B. , Sahm H.
Published: December 2004

Using permeabilized Zymomonas mobilis or glucose–fructose oxidoreductase isolated from this microorganism a model system for biosensors with a protein confined NADP(H) cofactor for the determination of glucose, fructose, gluconolactone, and sorbitol was developed. Either permeabilized microorganisms containing the oxidoreductase or the pure enzyme were confined via membrane separation in a small measuring chamber, that was integrated into a flow injection analysis system (FIA). The measuring principle was the monitoring of the NAD(P)H fluorescence, excited at 360 nm and measured at 450 nm. NADP(H), which is confined in the protein complex, was oxidized or reduced during the enzymatic reactions and the changes in the fluorescence intensity were related to the substrate concentration. The sensitivity of the system covered a range from 0.001 to 100 g/L of the analyte depending on substrate and operating conditions. The applicability of this model system for bioprocess monitoring was proved using samples from a Pseudomonas pseudoflava cultivation. © 1993 John Wiley & Sons, Inc.
A thermodynamically based correlation for maintenance gibbs energy requirements in aerobic and anaerobic chemotrophic growth
Authors: Tijhuis L. , Van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

A thermodynamic framework has been provided for the description of maintenance requirements of microorganisms. The central parameter is the biomass specific Gibbs energy consumption for maintenance, mE (kJ/C‐mol biomass · h). A large set of data has been used including (i) a large range of different organisms (bacteria, yeasts, plant cells), (ii) mixed cultures, (iii) heterotrophic and autotrophic growth, (iv) growth under aerobic and anaerobic conditions, and (v) a large temperature range (5–75°C). It appears that only the temperature has a major influence, with an energy of activation of 69 kJ/mol. Different electron donors or electron acceptors only show a very minor influence on mE. On the basis of the data set, temperature correlations of mE have been derived for aerobic and anaerobic growth. The generalized concept for maintenance Gibbs energy is used to establish a correlation which allows the estimation of the biomass yield on electron donor as a function of C‐source, electron donor, electron acceptor, N source, growth rate, and temperature. The advantage of using the mE parameter over other maintenance‐related parameters (like μe, mO2, mD, γDmD) is discussed. © 1993 John Wiley & Sons, Inc.
On‐line monitoring of cell concentration of Perilla frutescens in a bioreactor
Authors: Zhong Jian‐Jiang , Fujiyama Kazuhito , Seki Tatsuji , Yoshida Toshiomi
Published: December 2004

This article demonstrates the successful in situ real‐time monitoring of the cell concentration of Perilla frutescens in a bioreactor by using a laser turbidimeter. It was found that turbidity measurements at 780 nm with the laser sensor were hardly affected by the red color of the anthocyanin produced by P. frutescens cells, nor by the aeration rate or agitation speed within the ranges investigated. There was an excellent linear relationship, with a correlation coefficient (r2) higher than 0.99, between the sensor's response and the cell concentration. The whole growth stage of the cells, i.e., lag, logarithmic, and stationary phases, in bioreactor cultivations, could be satisfactorily estimated on‐line by means of the in situ turbidimeter. However, during the declining phase of the cells, an apparent deviation was observed between the on‐line estimations and off‐line measurements of cell concentrations by dry cell weight, while the wet cell weight could be estimated by the same turbidimeter system. We found that this deviation was caused by a decrease in the cell density due to an increase of the individual cell volume and a decrease of the cell dry weight during the declining phase. © 1993 John Wiley & Sons, Inc.
Use of a simple mathematical model to predict the behavior of Escherichia coli overproducing β‐lactamase within continuous single‐ and two‐stage reactor systems
Authors: Togna A. Paul , Fu Jeffrey , Shuler Michael L.
Published: December 2004

A simple mathematical model is developed to help explain the complex population dynamics of an Escherichia coli host–plasmid expression/excretion system for β‐lactamase within single‐ and two‐stage reactors. The model successfully integrates the individual regulatory (tac promoter induction), genetic (runaway plasmid replication), and population dynamics (culture instability) aspects of the system. The model predicts, and experiment confirms, that high‐level β‐lactamase production and excretion cannot be easily maintained in single‐stage reactors using the current plasmid construction. Stable target protein production and excretion is mathematically predicted, and experimentally confirmed, within two‐stage reactors. The model is used to provide insight into engineering a more stable host–vector expression/excretion system for use in single‐stage reactors. © 1993 John Wiley & Sons, Inc.
Optimization of hepatocyte attachment to microcarriers: Importance of oxygen
Authors: Foy Brent D. , Lee Jaegwan , Morgan Jeff , Toner Mehmet , Tompkins Ronald G. , Yarmush Martin L.
Published: December 2004

Many potential applications of primary hepatocytes cultured on microcarriers, such as an artificial liver or hepatocyte transplantation, would benefit from having a large number of hepatocytes attached to each microcarrier. In addition, the supply of primary hepatocytes is usually limited, so the efficient utilization of hepatocytes during attachment to microcarriers is necessary. Several physical parameters involved in the attachment process have been investigated, and the number of cells attached per microcarrier and the fraction of hepatocytes which attach have been quantitatively monitored. Variation of the partial pressure of gas phase oxygen in the incubation flask produced significant effects on the attachment of hepatocytes to microcarriers, with higher partial pressures of oxygen found to be necessary for attachment. In addition, variation of fluid depth and cell number, both of which influence the partial pressure of oxygen at the cell surface, affected hepatocyte attachment. The partial pressure of oxygen at the cell surface as a function of the physical parameters was analyzed using a simple one‐dimensional theoretical model. Variations in the cell‐to‐microcarrier ratio used for incubation indicate that a compromise must be made in terms of maximizing the number of cells per microcarrier and the fraction of total hepatocytes which attach. The maximum number of hepatocytes per microcarrier obtained in this work was approximately 100. The best attachment fraction, defined as the ratio of the number of hepatocytes attached to the total number added to the incubation, was approximately 90%. © 1993 John Wiley & Sons, Inc.
Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase
Authors: Hedström Gun , Backlund Maria , Slotte J. Peter
Published: December 2004

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water‐in‐oil microemulsion (AOT/isooctane). By using n‐propanol as the alcohol, an optimal W0 ([H2O]/[AOT] ratio) of 12 was found for the synthesis of n‐propyl‐ibuprofenate at room temperature. The lipase showed high preference for the S(+)‐enantiomer of ibuprofen, which was esterified to the corresponding S(+)‐ibuprofen ester. The R(−)‐ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S‐ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n‐alcohols with different chain lengths (3–12) were compared, and it appeared that short‐ (propanol and butanol) and long‐chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed to the corresponding S‐ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S‐ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S‐ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due to interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems. © 1993 John Wiley & Sons, Inc.
Prevascularization of porous biodegradable polymers
Authors: Mikos Antonios G. , Sarakinos Georgios , Lyman Michelle D. , Ingber Donald E. , Vacanti Joseph P. , Langer Robert
Published: December 2004

Highly porous biocompatible and biodegradable polymers in the form of cylindrical disks of 13.5 mm diameter were implanted in the mesentery of male syngeneic Fischer rats for a period of 35 days to study the dynamics of tissue ingrowth and the extent of tissue vascularity, and to explore their potential use as substrates for cell transplantation. The advancing fibrovascular tissue was characterized from histological sections of harvested devices by image analysis techniques. The rate of tissue ingrowth increased as the porosity and/or the pore size of the implanted devices increased. The time required for the tissue to fill the device depended on the polymer crystallinity and was smaller for amorphous polymers. The vascularity of the advancing tissue was consistent with time and independent of the biomaterial composition and morphology. Poly(L‐lactic acid) (PLLA) devices of 5 mm thickness, 24.5% crystallinity, 83% porosity, and 166 μm median pore diameter were filled by tissue after 25 days. However, the void volume of prevascularized devices (4%) was minimal and not practical for cell transplantation. In contrast, for amporphous PLLA devices of the same dimensions, and the similar porosity of 87% and median pore diameter of 179 μm, the tissue did not fill completely prevascularized devices, and an appreciable percentage (21%) of device volume was still available for cell engraftment after 25 days of implantation. These studies demonstrate the feasibility of creating vascularized templates of amorphous biodegradable polymers for the transplantation of isolated or encapsulated cell populations to regenerate metabolic organs and tissues. © 1993 John Wiley & Sons, Inc.
Removal of lead from aqueous solutions by Penicillium biomass
Authors: Niu Hui , Xu Xue Shu , Wang Jian Hua , Volesky B.
Published: December 2004

The removal of lead ions from aqueous solutions by adsorption on nonliving Penicillium chrysogenum biomass was studied. Biosorption of the Pb+2 ion was strongly affected by pH. Within a pH range of 4 to 5, the saturated sorption uptake of Pb+2 was 116 mg/g dry biomass, higher than that of activated charcoal and some other microorganisms. At pH 4.5, P. chrysogenum biomass exhibited selectivity for Pb+2 over other metal ions such as Cd+2, Cu+2, Zn+2, and As+3 Sorption preference for metals decreased in the following order: Pb > Cd > Cu > Zn > As. The sorption uptake of Pb+2 remained unchanged in the presence of Cu+2 and As+3, it decreased in the presence of Zn+2, and increased in the presence of Cd+2. © 1993 John Wiley & Sons, Inc.
Self‐cycling fermentation in a stirred tank reactor
Authors: van Walsum G. Peter , Cooper David G.
Published: December 2004

Self‐cycling fermentations (SCFs) were conducted in a stirred tank apparatus using Bacillus subtilis and Acinetobacter calcoaceticus. The systems were very stable and the experiments lasted through many cycles. The variation of parameters such as biomass and doubling time from cycle to cycle was small. The stirred tank reactor (STR) allowed a much better control of the working volume in the fermentor from cycle to cycle, compared to the cyclone column, and it was not necessary to make periodic corrections.

The production of surfactin from B. subtilis was achieved without extending the cycle time. The harvested broth at the end of each cycle was allowed to remain in a secondary vessel, at ambient temperature, before being collected. It is exhaustion of the limiting nutrient which causes an increase in dissolved oxygen (DO). At this point, the computer, which constantly monitors the DO, triggered the harvesting sequence to end the cycle. Thus, the mature culture in the secondary vessel experienced appropriate conditions for the production of the secondary metabolite. Meanwhile, the next batch of cells was being grown in the primary reactor.

The response of a gas analyzer on the effluent paralleled that of the DO measurements in the fermentor. These data for oxygen and carbon dioxide exhibited less noise than the DO readings. Either would be a more reliable parameter for feedback control of the SCF because the problem of fouling of the DO probe after extended runs of many cycles would be eliminated. © 1993 John Wiley & Sons, Inc.
Real‐Time compensation of the inner filter effect in high‐density bioluminescent cultures
Authors: Konstantinov Konstantin B. , Dhurjati Prasad , Van Dyk Tina , Majarian William , Larossa Robert
Published: December 2004

Bioluminescence has recently become a popular research tool in several fields, including medicine, pharmacology, biochemistry, bioprocessing, and environmental engineering. Beginning with purely qualitative goals, scientists are now targeting more demanding applications where accurate, quantitative interpretation of bioluminescence is necessary. Using the recent advances in fiber‐optic technology, bioluminescence is easily monitored in vivo and in real time. However, the convenience of this measurement is often concealing an unsuspected problem: the bioluminescence signal might be corrupted by a large error caused by the extinction of light by biological cells. Since bioluminescent cultures not only emit light but also absorb and scatter it, the measured signal is related in a complex, nonlinear, and cell‐concentration‐dependent manner to the “true” bioluminescence. This light extinction effect, known as the “inner filter effect,” is significant in high‐density cultures. Adequate interpretation of the bioluminescence signal can be difficult without its correction. Here, we propose a real‐time algorithm for elimination of the inner filter effect in a bioreactor. The algorithm yields the bioluminescence which would be measured if the glowing culture was completely transparent. This technique has been successfully applied to batch and continuous cultivation of recombinant bioluminescent Escherichia coli. © 1993 John Wiley & Sons, Inc.
Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors
Authors: Gikas P. , Livingston A. G.
Published: December 2004

This work describes investigations into the viability of cells growing on 3,4‐dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2‐L working volume, and a three‐phase air lift (TPAL) bioreactor with a 3‐L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h−1 in the CST bioreactor and between 0.111 and 0.500 h−1 in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris‐EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass‐specific ATP concentration increases from 0.44 to 1.86 mg ATP g−1 dry weight (dw) as dilution rate increases from 0.027 to 0.115 h−1. At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g−1 dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g−1 dw at dilution rates between 0.111 and 0.200 h−1 to approximately 0.119 mg ATP g−1 dw at dilution rates between 0.300 and 0.500 h−1. This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. © 1993 John Wiley & Sons, Inc.
Differences in response of Zymomonas mobilis and Saccharomyces cerevisiae to change in extracellular ethanol concentration
Authors: Hobley T. J. , Pamment N. B.
Published: December 2004

In high cell density batch fermentations, Zymomonas mobilis produced 91 g L−1 ethanol in 90 min but culture viability fell significantly. Similar viability losses in rapid fermentations by yeast have recently been shown to be attributable in part to the high rate of change of the extracellular ethanol concentration. However, in simulated rapid fermentations in which ethanol was pumped continuously to low cell density Z. mobilis suspensions, increases in the rate of change of ethanol concentration in the range 21–83 g L−1 h−1 did not lead to accelerated viability losses. The lag phase of Zymomonas cultures exposed to a 30‐g L−1 step change in ethanol concentration was much shorter than that of Saccharomyces cerevisiae, providing evidence that the comparative insensitivity of Zymomonas to high rates of change of ethanol concentration is due to its ability to adapt to changes in ethanol concentration more rapidly than yeast. © 1994 John Wiley & Sons, Inc.
Diffusivity of Cu2+ in calcium alginate gel beads
Authors: Jang Larry K.
Published: December 2004

The diffusivity of Cu2+ in calcium alginate beads calculated by the shrinking core model (SCM) was reevaluated in this work. The results obtained in this work were significantly different than those by the original authors. There were excellent agreements between the results obtained by the SCM in this work and those by the more rigorous linear absorption model (LAM) by the original authors. © 1994 John Wiley & Sons, Inc.
Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A
Authors: Wilson S.‐A. , Peek K. , Daniel R. M.
Published: December 2004

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG‐immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m2 of support. Saturation of the CPG beads was observed at 540 azoU/m2 of 105‐nm beads. Lower levels (50 azoU/m2 of 50‐nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80°C increased on immobilization at all pH values between 5 and 11, the greatest increase in half‐life being approximately 12‐fold at pH 7.0. Temperature–activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl2. Overall, the results show that low levels of calcium (10 μM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. © 1994 John Wiley & Sons, Inc.
Two‐Step biocatalytic conversion of an ester to an aldehyde in reverse micelles
Authors: Yang Fangxiao , Russell Alan J.
Published: December 2004

Lipases from Candida cyclindracea (L‐1754) and wheat germ (L‐3001) have been used to hydrolyze esters to their corresponding alcohols and acids in reverse micelles. Alcohol dehydrogenase from baker's yeast (YADH) was subsequently used to reduce the alcohol products to aldehydes. Cofactor recycling in the redox reaction was achieved using a sacrificial cosubstrate, as described previously. Four surfactants (sodium dioctylsulfosuccinate, Nonidet P‐40 with Triton X‐35, polyoxyethylene, 10‐cetyl‐ether, polyoxyethylene sorbitan trioleate) were employed to determine the effect of amphiphile on ester hydrolysis and redox reaction rates separately. The effect of type of organic solvent, W0 [(water]/[surfactant)], and substrate concentration on separte enzyme activity were also investigated. A brief investigation of a single phase, two‐step reaction catalyzed by the combination of lipase and YADH in reverse micelles is also reported. The activities of the enzymes are significantly different when used together instead of independently. © 1994 John Wiley & Sons, Inc.
Separation of penicillin G from phenylacetic acid in a supported liquid membrane system
Authors: Lee Chan‐Jen , Yeh Huey‐Jiuan , Yang Wu‐Yung , Kan Chin‐Rang
Published: December 2004

The separation of penicillin G (Pen G) from phenylacetic acid (PAA) by use of a supported liquid membrane (SLM) system with Amberlite LA‐2 dissolved in 1‐decanol, supported on a microporous polypropylene membrane, was studied. The results show that the individual permeability of each component in mixture was lower than that in a single compartment system and, it suggests a strong transport competition between Pen G and PAA. The SLM system in this study proved to be a promising process for the selective separation of Pen G from PAA. The maximum separation factor was found to be 1.8 under a liquid membrane resistance controlled mechanism. © 1994 John Wiley & Sons, Inc.
Purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography
Authors: Wang Min‐Ying , Bentley William E. , Vakharia Vikram
Published: December 2004

Over the past 10 years, the baculovirus–insect cell system has become a powerful and versatile tool for the expression of a variety of heterologous proteins. In order to simplify separation of a cloned protein from the baculovirus–insect expression system, we have cloned a gene encoding for the protein of interest, a structural protein (VP2) of a strain (E/DEL) of infectious bursal disease virus (IBDV), with a metal ion binding site (His)5 at its C‐terminus. This chimeric protein (VP2H) has been expressed and one‐step affinity purified with immobilized metal ions (Ni+2). With antigen capture–enzyme‐linked immunosorbent assay (AC‐ELISA), we determined that the conformation of this chimeric protein was no different from the recombinant wild‐type VP2 protein. However, the two proteins (VP2 and VP2H) can be distinguished and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and detected immunologically following Western blotting. © 1994 John Wiley & Sons, Inc.
Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface
Authors: Jordan M. , Eppenberger H. M. , Sucker H. , Widmer F. , Einsele A.
Published: December 2004

We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration‐dependent and height‐dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium–bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. © 1994 John Wiley & Sons, Inc.
Model of the anaerobic metabolism of the biological phosphorus removal process: Stoichiometry and pH influence
Authors: Smolders G. J. F. , van der Meij J. , van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

In the anaerobic phase of a biological phosphorus removal process, acetate is taken up and converted to PHB utilizing both energy generated in the degradation of polyphosphate to phosphate, which is released, and energy generated in the conversion of glycogen to poly‐β‐hydroxy butyrate (PHB). The phosphate/acetate ratio cannot be considered a metabolic constant, because the energy requirement for the uptake of acetate is strongly influenced by the pH value. The observed phosphate/acetate ratio shows a variation of 0.25 to 0.75 P‐mol/C‐mol in a pH range of 5.5 to 8.5. It is shown that stored glycogen takes part in the metabolism to provide reduction equivalents and energy for the conversion of acetate to PHB. A structured metabolic model, based on glycogen as the source of the reduction equivalents in the anaerobic phase and the effect of the pH on the energy requirement of the uptake of acetate, is developed. The model explains the experimental results satisfactorily. © 1994 John Wiley & Sons, Inc.
Studies on microbial chromate reduction by Pseudomonas Sp. In aerobic continuous suspended growth cultures
Authors: Gopalan R. , Veeramani H.
Published: December 2004

Reduction of hexavalent chromium was studied in three bench‐scale continuous stirred tank reactors. The inoculum was a culture of Pseudomonas sp., capable of giving 83% to 87% chromate reduction in 72‐h batch assays with 60 mg Cr(VI) L−1 in synthetic medium. The continuous culture studies were conducted for about 100 days using synthetic feed containing different levels of chromate (5 to 124 mg L−1) at 28° to 30°C and pH 6.8. The feed rate was varied over the range 0.5 to 1 L d−1 to obtain hydraulic retention time of 36 to 72 h. Chromate reduction efficiency was 81% to 91% and 100% for influent Cr(VI) concentrations of 15 to 124 and 5 mg L−1, respectively, with a hydraulic retention time of 72 h. © 1994 John Wiley & Sons, Inc.
Cadmium removal in a biosorption column
Authors: Volesky B. , Prasetyo I.
Published: December 2004

New biosorbent material derived from a ubiquitous brown marine alga Ascophyllum nodosum has been examined in packed‐bed flow‐through sorption columns. It effectively removed 10 mg/L of cadmium down to 1.5 ppb levels in the effluent, representing 99.985% removal. The experimental methodology used was based on the early Bohart and Adams sorption model, resulting in quantitative determination of the characteristic process parameters which can be used for performance comparison and process design. An average metal loading of the biosorbent (N0) determined was 30 mg Cd/g, corresponding closely to that observed for the batch equilibrium metal concentration of 10 mg Cd/L. The critical bed depth (Dmin) for the potable water effluent quality standard (0.005 mgg Cd/L) varied with the column feed flow rate (2.4 to 9.6 L/h · cm2) from 20 to 50 cm. The sorption column mass transfer and dispersion coefficients were determined, which are also required for solving the sorption model equations. © 1994 John Wiley & Sons, Inc.
Enzymatic synthesis of alkyl β‐D‐xylosides by transxylosylation and reverse hydrolysis
Authors: Drouet Philippe , Zhang Mu , Legoy Marie Dominique
Published: December 2004

TheTrichoderma reeseiβ‐xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl β‐D‐xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl β‐D‐xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium‐controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium‐controlled synthesis. The specificity of the β linkage is confirmed by proton nuclear magnetic resonance (1H NMR) analysis. © 1994 John Wiley & Sons, Inc.
Communication to the editor. Application of the cross‐regulation system as a metabolic switch
Authors: Chen Wilfred , Bailey James E.
Published: December 2004

The ability to switch metabolic flow from one pathway to another at a desired point in a bioprocess expands the horizons of metabolic engineering. Such an externally inducible switch can be realized by embedding synthetic operons behind tow corss‐regulated promoters. This results in coordinated cessation of transcription of one operon while transcription of a second operon is simultaneously activated. The ability to effect such coordinated and inverse control of transcription of two operons has been illustrated experimentally using a model construct containing two different reporter genes, Vitreoscilla hemoglobin (VHb) and chloramphenicol acetyltransferase (CAT), fused to λPL and tac promoters, respectively, along with corresponding repressor genes in a cross‐regulation configuration. Only VHb production was observed preinduction, and postinduction only CAT was produced. The framework presented here and its obvious extensions can be used with different combinations of promoter systems and synthetic operon constructs to achieve complicated metabolic flux regulation in diverse host. © 1994 John Wiley & Sons, Inc.