bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of compost biofiltration system degrading dichloromethane
Authors: Ergas Sarina. J. , Kinney Kerry , Fuller Mark E. , Scow Kate M.
Published: December 2004

The effects of acclimatization of microbial populations, compound concentration, and media pH on the biodegradation of low concentration dichloromethane emissions in biofiltration systems was evaluated. Greater than 98% removal efficiency was achieved for dichloromethane at superficial velocities from 1 to 1.5 m3/m3. min (reactor residence times of 1 and 0.7 min, respectively) and inlet concentrations of 3 and 50 ppm Although acclimatization of microbial populations to toluene occurred within 2 weeks of operation start‐up, initial dichloromethane acclimatization took place over a period of 10 weeks. This period was shortened to 10 days when a laboratory grown consortium of dichloromethane degrading organism, isolated from a previously acclimatized column, was introduced into fresh biofilter media. The mixed culture consisted to 12 members, which together were able to degrade dichloromethane at concentrations up to 500 mg/L. Only one member of the consortium was able to degrade dichloromethane were sustained for more than 4 months in a biofilter column receiving an inlet gas stream with 3 ppmv of dichloromethane acidification of the column and resulting decline in performance occurred when a 50‐ppmv inlet concentration was used. A biofilm model incorporating first order biodegradation kinetics provided a good fit to observed concentration profiles, and may prove to be a useful tool for designing biofiltration systems for low concentration VOC emissions. © 1994 John Wiley & Sons, Inc.
Induction of apoptosis in nutrient‐deprived cultures of hybridoma and myeloma cells
Authors: Mercille Sylvain , Massie Bernard
Published: December 2004

In the present study, cell death was investigated in cultures of NS/0 myelomas and SP2/0‐derived D5 hybridomas through morphological examination of cells stained with acridine orange and ethidium bromide. The relative contribution of elevated levels of lactic acid and ammonia, as well as deprivation of glutamine, cystine, and glucose on the induction of necrosis or apoptosis, was investigated. In batch culture of D5 hybridoma cells, induction of apoptotic cell death correlated with the exhaustion of glutamine, while in the case of NS/0 myelomas, it coincided with exhaustion of cystine. To determine whether limiting nutrients were the actual triggering factors for apoptosis in batch culture, exponentially growing cells were resuspended in glutamine or cystine‐free media. Within 30 to 40 h, viability decreased to 50% and the nonviable cell population displayed typical apoptotic morphology, with crescents of condensed chromatin around the periphery of the nucleus, or with the entire nucleus present as one or a group of featureless, brightly staining spherical beads. Similarly, D5 hybridomas and NS/0 myelomas cultivated in glucose‐free medium died mainly from apoptosis. Cells were also cultivated in fresh medium supplemented with elevated concentrations of ammonia (3.0 mM) and/or lactate (35 mM, 50 mM). This resulted in decreased viabilities and necrotic death in both cell lines. From these results, we conclude that D5 hybridomas and NS/0 myelomas deprived of essential nutrients die by apoptosis, whereas incubation in the presence of elevated levels of metabolic byproducts such as ammonia and lactate will induce necrotic cell death in these cells. © 1994 John Wiley & Sons, Inc.
Physiological responses of hybridoma cells in a protein‐free medium to soluble and immobilized antigen
Authors: Dandulakis Gregory , Herr John C. , Kirwan Donald J.
Published: December 2004

This study examined the effect of antigen in a protein free medium on cell growth and monoclonal antibody production by a hybridoma line. Antigen immobilized on a Sepharose gel matrix via a bovine γ‐globulin carrier protein was used to stimulate the cell cultures in T‐flasks. In comparison to antigen‐free culture, total antibody production during was increased up to 40%, while slower cell growth rates were observed. The specific antibody production during the stationary culture phase was 40% to 80% higher in the presence of immobilized antigen. The surface density of antigen on the Sepharose beads had a strong influence on the physiological response of the hybridomas. © 1994 John Wiley & Sons, Inc.
Membrane bioreactor with a porous hydrophobic membrane as a gas–liquid contactor for waste gas treatment
Authors: Reij Martine W. , de Bont Jan A. M. , Hartmans Sybe , de Gooijer Kees D.
Published: December 2004

A novel type of bioreactor for waste gas treatment has been designed. The reactor contains a microporous hydrophobic membrane to create a large interface between the waste gas and the aqueous phase. To test the new reactor, propene was chosen because of its high air/water partition coefficient, which causes a low water concentration and hampers its removal from air. Propene transfer from air to a suspension of propene‐utilizing Xanthobacter Py2 cells in the membrane bioreactor proved to be controlled by mass transfer in the liquid phase. The resistance of the membrane was negligible. Simulated propene transfer rates agreed well with the experimental data. A stable biofilm of Xanthobacter Py2 developed on the membrane during prolonged operation. The propene flux into the biofilm was 1 × 10−6 mol m−2 s−1 at a propene concentration of 9.3 × 10−2 mol m−3 in the gas phase. © 1995 John Wiley & Sons, Inc.
On‐line calibration of a computerized biosensor system for continuous measurements of glucose and lactate
Authors: Kyröläinen Marika , Håkanson Håkan , Mattiasson Bo
Published: December 2004

An on‐line calibration procedure for application in continuous monitoring systems has been developed. Control of the calibration value and recalibration on‐line during monitoring is possible without having to disrupt the sample withdrawal. The calibration procedure has been applied and evaluated in a continuous biosensor system based on the detection of oxygen depletion during enzymatic substrate conversion by immobilized oxidases. Evaluation included on‐line calibration during continuous measurements of glucose and lactate in bovine blood samples. Calibration of the complete system consisting of a sampling device, a sample handling step, a biocatalytic step, a detection step, and a data processing unit is performed by the on‐line addition of a calibration solution to a blank sample which is fed through the system. The calibration cycle is completed within 5.5 min. When recalibration is carried out during monitoring, the calibration solution is added to the sample, instead of to a blank sample, and the increase in outlet singl is registered. The major advantage of this internal standard principle is that the calibration solution is fed through the whole system according to the same path as the sample solution and thus takes into account all parameters influencing the sample. © 1995 John Wiley & Sons, Inc.
Influence of water activity on the synthesis of triolein catalyzed by immobilized Mucor miehei lipase
Authors: Dudal Yves , Lortie Robert
Published: December 2004

The influence of the thermodynamic activity of water (aw)on the synthesis of triolein catalyzed by Mucor miehei lipase was investigated. Its effect on the equilibrium and on the rates of the different reactions present, esteification and mono‐ and diglyceride isomerization, was evaluated through measurements made in controlled water activity atmosphere. The apparent equilibrium constants were measured from the concentration of the different species as a function of the intial glycerol‐to oleic‐acid ratio using all the values at once with a multi‐response nonlinear regression technique. Rate constants were determined from kinetic measurements and non‐linear regression uning the variation of the concentration of all significant species in the system. Except for the synthesis of diolein from monoolein, which shows a maximum for aw ≈ 0.5, the apparent rate constants of the various reactions are not significantly affected by the value of the water activity. The equilibrium is shifted to‐ward the synthesis of triolein for low values of aw, indicating that in the design of a process for triglyceride synthesis, using M. miehei lipase as a catalyst, the water activity can be lowered to extreme values to favor the synthesis, without any sacrifice on the productivity of the process. © 1995 John Wiley & Sons, Inc.
Serum‐free cell culture on insulin‐immobilized porous collagen beads
Authors: Ito Yoshihiro , Zheng Ji , Imanishi Yukio
Published: December 2004

Insulin or albumin was immobilized on collagen beads using water‐soluble carbodiimide. Adhesion of STO mouse fibroblast cells onto the beads decreased with increasing the amount of immobilized proteins. Growth of the cells was remarkably accelerated on the insulinimmobilized collagen beads, which can be used for serum‐free cell culture. The growth acceleration became larger with increasing the amount of immobilized insulin, while it became smaller with increasing the amount of immobilized albumin. In addition, the immobilized insulin more strongly accelerated the cell growth than free insulin plus collagen beads. © 1995 John Wiley & Sons, Inc.
Death mechanisms of animal cells in conditions of intensive agitation
Authors: Al‐Rubeai M. , Singh R. P. , Goldman M. H. , Emery A. N.
Published: December 2004

The question is addressed as to whether cells which are subject to high‐energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench‐scale (1‐L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m−3 there was no apparent damage. At 320 W m−3 cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m−3 in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller‐sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F‐actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate. © 1995 John Wiley & Sons, Inc.
A modeling study of anaerobic biofilm systems: I. Detailed biofilm modeling
Authors: Flora Joseph R. V. , Suidan Makram T. , Biswas Pratim , Sayles Gregory D.
Published: December 2004

A detailed model acetate‐utilizing methanogenic biofilms accounting for the diffusion of neutral and ionic species, chemical equilibrium, electroneutrality, gas production within the biofilm, pH‐dependent Monod kinetics, and the presence of a concentration boundary layer is presented. The model qualitatively fits the pH profiles that are reported for acetate‐utilizing methanogenic aggregates. A sensitivity analysis on the biological parameters showed that the flux of acetate is sensitive to the maximum utilization rate, half‐saturation constant, and biofilm density for the bulk conditions investigated. Criteria when traditional biofilm models can be used to predict the flux of acetate into the biofilm are established. If the maximum pH change predicted using a hypothetical system is within ±0.05, the traditional model predicts the flux to within ±5% of the value calculated with the model developed in this study. © 1995 John Wiley & Sons, Inc.
Degradation of quinoline by immobilized Comamonas acidovorans in a three‐phase airlift reactor
Authors: Ulonska A. , Deckwer W.‐D. , Hecht V.
Published: December 2004

Quinolie degradation by Comamonas acidovorans was studied in a continuously operated three‐phase airlift reactor. Porous glass beads were applied as support matrix for cell imobilization by colonization. Under steady‐state conditions (S ∼ 0), cell attachment was poor at low dilution rates but imporved considerably with increasing dilution rate. Conversion of quinoline was investigated below and above the washout for suspended culture (Dcrit = μmax = 0.42 h−1). With immobilized cells the reactor could be operated at D > μmax, and complete conversion of quinoline was achieved as long as the specific quinoline feed rate D*S0/X did not exceed the maximum specific degradation rate (rS, max). The biofilm thickness was about 100 μm, and its efficiency was about 54% compared to suspended organisms. If quinoline overloads were supplied to the reactor, quinoline, as overloads were supplied to the reactor, quinoline, as well as its pathway intermediates, appeared in the reactor and conversion was low. Hence, the immobilized microorganisms remained viable and active. They could survive quinoline overloads. If the quinoline feed rate was reduced agains, complete conversion was reestablished. © 1995 John Wiley & Sons, Inc.
Photochemistry of a photosynthetic reaction center immobilized in lipidic cubic phases
Authors: Hochkoeppler Alejandro , Landau Ehud M. , Venturoli Giovanni , Zannoni Davide , Feick Reiner , Luisi Pier Luigi
Published: December 2004

Photosynthetic reaction centers, isolated and purified from the facultative phototrophic bacterium Chloroflexus aurantiacus, were immobilized in optically transparent lipidic cubic phases composed of 42% (w/w) 1‐palmitoyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine and 58% (w/w) water. The immobilized photosynthetic protein retains its native properties, as indicated by visible and circular dichroic spectra. The ground state visible spectrum of the immobilized reaction centers is very similar to the corresponding spectrum in aqueous solution, indicating that the protein pigments are not extracted into the lipidic regions of the cubic phase. The secondary structure of the protein is maintained in the immobilized state, as determined by far‐UV circular dichroism spectroscopy in the 200‐ to 250‐nm range. Moreover, immobilized reaction centers retain their photochemical activity: a reversible photo‐oxidation of the primary electron donor (P) is seen upon continuous illumination. Furthermore, the entrappment of reaction centers does not affect the kinetics of charge recombination between the photo‐oxidized primary donor (P+) and the photoreduced primary quinone acceptor, generated by a short flash of light. Reaction centers devoided of the secondary quinone acceptor can be easily reconstituted in cubic phases by means of their coimmobilization with 1,4‐naphtoquinone. Indeed, the kinetics for charge recombination in reconstituted reaction centers is dramatically slower than the corresponding kinetics in the unreconstituted protein. Interestingly, immobilized reaction centers are significantly stabilized as compared with reaction centers in aqueous solution: the integrity of the protein in the cubic phase is maintained for at least 5 months, whereas in water solution 50% of the activity is lost within 2 months. © 1995 John Wiley & Sons, Inc.
Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon‐γ
Authors: Coppen Steven R. , Newsam Ray , Bull Alan T. , Baines Anthony J.
Published: December 2004

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon‐γ (IFN‐γ), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN‐γ. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN‐γ within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN‐γ are heterogeneous in their environment, with variable access to O2 and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell–cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell–cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. © 1995 John Wiley & Sons, Inc.
The oxidation of chiral alcohols catalyzed by catalase in organic solvents
Authors: Magner Edmond , Klibanov Alexander M.
Published: December 2004

The catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks down tert‐butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase‐catalyzed production of tert‐butanol from tert‐butyl hydroperoxide increases more than 400‐fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate‐limiting step of the enzymatic process involves the reduction of catalase's compound I by tert‐butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols with tert‐butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3‐butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3‐butanediol in a series of organic solvents reveals a considerable solvent dependence. © 1995 John Wiley & Sons, Inc.
A cometabolic kinetics model incoroporating enzyme inhibition, inactivation, and recovery: II. Trichloroethylene degradaation experiments
Authors: Ely Roger L. , Hyman Michael R. , Arp Daniel J. , Guenther Ronald B. , Williamson Kenneth J.
Published: December 2004

A Cometabolism enzyme kinetics model has been presented which takes into account changes in bacterial activity associated with enzyme inhibitiion, inactivation, inactivation of enzyme resulting from product toxicty, and respondent synthesis of new enzyme. Although this process is inherently unsteady‐state, the model assumes that cometabolic degradation of a compound exhibiting product toxicity can be modeled as pseudo‐steady‐staate under certain conditions. In its simplified from, the model also assumes that enzyme inactivation is directly propoertional to nongrawth substrate oxidation, and that recovery is directly proportionla to growth substrate oxidation. In part 1, model drivation, simplification, and analyses were described. In this articles, model assuptiions are tested by analyzing data from experiments exmining trichloroethylene (TCE) degradation by the ammoniaoxidizing baceterium Nitrosomonas europaea in a quasisteady‐state bioreactor. Model solution results showed steady‐state bioreactor. Model solution results showed TCE to be a competitive inhibitoer of ammonia oxidation, with TCE affinity for ammonia monooxygenase (AMO) being about four times greater than that of ammonia for the enzyme. Inhibition was independent fo TCE oxidation and occurred essentially instantly upon exposure to TCE. In contrast, inactivation of AMO occurred more gradually and was proportional to the rate and amount of TCE oxidized. Evaluation of other O2‐dependent enzymes and electron transport proteins suggested that TCE‐related damage was predominantly confined to AMO. In response to inhibition and/or inactivation, bacterial recovery was initiated, even in the presence of TCE, implying that membranes adn protein synthesis systems were functioning. Analysis of data and comparison of model results showed the inhibition/inactivation/recovery concept to provide a reasonable basis for understandign the effects fo TCE on AMO function and bacterial response. The model assumptions were verified except tht questions remain regarding the factores controlling recovery and its role in the long term. © 1995 John Wiley & Sons, Inc.
Detachment of biomass from suspended nongrowing spherical biofilms in airlift reactors
Authors: Gjaltema A. , Tijhuis L. , van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

In three‐phase internal loop airlift reactors, the detachment of biomass from suspended biofilm pellets in the presence of bare carrier particles was investigated under nongrowth conditions. The detachment rate was dominated by collisions between bare carrier particles and biofilm pellets. The concentration of bare carrier particles and the carrier roughness strongly influenced the detachment rate. A change in flow regime from bubbling to slug flow considerably increased the detachment rate. Otherwise, the superficial gas velocity did not directly affect the detachment rate. The influence of particle size was not clear. The bottom clearance did not affect the detachment rate within the tested range. Other aspects of reactor geometry might be important. The main detachment processes were abrasion and breakage of biofilm pellets. During the detachment process, two phases could be distinguished. In the first phase the detachment was relatively high, and both breakage and abrasion of biofilm pellets occurred. During the second phase, breakage dominated and the detachment rate was lower. The two‐phase behavior is explained by differences in strength between the inner and outer biofilm layers, possibly caused by variations in local growth rates during biofilm formation. Differences in growth history might also explain the various detachment rates observed with different biofilm batches. © 1995 John Wiley & Sons, Inc.
Propane‐Induced biodegradation of vapor phase trichoroethylene
Authors: Wilcox Donnell W. , Autenrieth Robin L. , Bonner James S.
Published: December 2004

Microbial degradation of trichloroethylene (TCE) has been demonstrated under aerobic conditions with propane. The primary objective of this research was to evaluate the feasibility of introducing a vapor phase form of TCE in the presence of propane to batch bioreactors containing a liquid phase suspension of Mycobacterium vaccae JOB5 to accomplish degradation. The reactor system consisted of three phases: a vapor phase introducing air, propane, and TCE; a liquid phase of the microbial suspension; and a solid phase in the form of the microorganisms. Long‐term and initial rate experiments were conducted on three culture sets to evaluate microbial response. In two long‐term test fed propane and approximately 0.1 mg/L and 1 mg/L of TCE, respectively, propane utilization was more efficient at the high TCE concentration (600 mmol propane/mmol TCE versus 11,900 mmol propane/mmol TCE), because the propane degradation rate was approximately the same for both tests (6.73 mg/L · h and 7.85 mg/L · h for the high and low tests). In addition, TCE utilization decreased after complete propane consumption. Initial rate tests on culture sets fed propane only revealed that cells with a history of exposure to a high concentration of TCE had the highest specific growth rate, but the lowest half‐saturation constant (7.60e−3 h−1 and 0.10 mg/L, respectively). Tests fed variable TCE concentrations (0.031 to 5.378 mg/L in the liquid phase) with no propane showed TCE depletion but no biomass growth. The tests revealed that the TCE removal increased as the TCE concentration increased, indicating a greater removal efficiency at the higher concentrations. Tests with a constant initial propane concentration and variable liquid phase TCE concentration revealed that specific propane utilization was essentially the same. © 1995 John Wiley & Sons, Inc.
Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities
Authors: Patnaik Ranjan , Spitzer Richard G. , Liao James C.
Published: December 2004

The synthesis of 3‐deoxy‐D‐arabino‐heptulosonate‐7‐phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during a condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4‐phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. This limitation can be relieved by (i) the recycling of pyruvate from PEP using PEP synthase (Pps) or (ii) use of non‐PTS sugars such as xylose. Previous studies have shown the usefulness of overexpressing tktA (encoding transketolase), aroG, and pps (PEP synthase) for DAHP production in an aroB strain unable to utilize DAHP further. In the present study we confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters. In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, as predicted by the stoichiometric analysis. However, this Pps effect is observed only in the presence of both increased AroG and TktA. In xylose mimimal medium, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown here are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.
Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media
Authors: Jönsson Åsa , Adlercreutz Patrick , Mattiasson Bo
Published: December 2004

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from −30° to 25°C. This article shows the kinetic reasons of this effect. α‐Chymotrypsin was immobilized on celite and used in four different water‐miscible solvents containing small amounts of water‐miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N‐acetyl‐L‐phenylalanine ethyl ester (Ac‐PheOEt) with L‐alaninamide (Ala‐NH2) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p‐values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N‐acetyl‐L‐phenylalanyl‐L‐ananinamide (AcPheAla‐NH2) as substrates. The Michaelis–Menten parameters, Km,app and Vmax, were determined for ester hydrolysis and dipeptide hydrolysis. Both Km,app and Vmax tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at −20%C compared with 84% at 25°C. © 1995 John Wiley & Sons, Inc.
An autoclavable glucose biosensor for microbial fermentation monitoring and control
Authors: Phelps Michael R. , Hobbs John B. , Kilburn Douglas G. , Turner Robin F. B.
Published: December 2004

The design, construction, and characterization of a prototype‐regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor‐indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed‐batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off‐line glucose analysis and was controlled manually at 0.44 ± 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high‐cell‐density fermentation. © 1995 John Wiley & Sons, Inc.
Biofilm parameters influencing biocide efficacy
Authors: Srinivasan Rohini , Stewart Philip S. , Griebe Thomas , Chen Ching‐I. , Xu Xiaoming
Published: December 2004

The influence of biofilm areal cell density, species composition, and the presence of abiotic particles on the disinfection and removal of bacterial biofilms by monochloramine was investigated. Mono‐ and binary population biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown on stainless‐steel slides in a continuous flow annular reactor. Biofilms were treated in the reactor with a pulse/step dose of 4 mg/L monochloramine for 2 h. Biofilm samples were disaggregated and assayed for colony formation on R2A agar and for total cell numbers by acridine orange direct counts. These data were used to determine apparent first order rate coefficients for the processes of disinfection and detachment. Disinfection rate coefficients exceeded detachment rate coefficients by as much as an order of magnitude and the two coefficients were poorly correlated (r = 0.272). The overall decay rate coefficient (disinfection plus detachment) depended strongly on the initial biofilm areal cell density. It displayed a parabolic dependence on cell density with a maximum near 108 cfu/cm2. This result points to multiple factors influencing biofilm susceptibility to antimicrobial challenge. Decay rates of K. pneumoniae measured in binary population biofilms were comparable with those measured in monopopulation biofilms (p = 0.61). P. aeruginosa decayed more slowly in biofilsm dominated by K. pneumoniae (p = 0.028), indicating some interaction between species. The presence of kaolin and calcium carbonate particles in the biofilm reduced disinfection efficacy. © 1995 John Wiley & Sons, Inc.
Synthesis of aspartame precursor with an immobilized thermolysin in mixed organic solvents
Authors: Miyanaga Masamitsu , Tanaka Takaaki , Sakiyama Takaharu , Nakanishi Kazuhiro
Published: December 2004

N‐(benzyloxycarbonyl)‐L‐aspartyl‐L‐phenylalanine methyl ester, a precursor of the synthetic sweetener, aspartame, was synthesized from N‐(benzyloxycarbonyl)‐L‐aspartic acid and L‐phenylalanine methyl ester with an immobilized thermolysin (EC 3.4.24.4) in the mixed organic solvent system of tert‐amyl alcohol and ethyl acetate. A mixed solvent consisting of tert‐amyl alcohol and ethyl acetate at a ratio of 33:67 (v/v) was found to be the most suitable with respect to synthetic rate and stability of the immobilized enzyme. The reaction continued to proceed quite successfully in a column reactor at 40°C and at a space velocity of 3.6 h−1 with a yield of 99%, using 40 mM Z‐Asp and 200 mM PheOMe dissolved in the mixed solvent as the substrate. © 1995 John Wiley & Sons, Inc.
Transport of lactate and acetate through the energized cytoplasmic membrane of Escherichia coli
Authors: Axe Douglas D. , Bailey James E.
Published: December 2004

Escherichia coli produces lactate and acetate in significant amounts during both aerobic and anaerobic glycolysis. A model describing the mechanism of protein mediated lactate transport has previously bee proposed. A simple theoretical analysis here indicates that the proposed model would be drain cellular energy resources by catalytically dissipating the proton‐motive force. An experimental analysis of lactate and acetate transport employ nuclear magnetic resonance (NMR) spectroscopy to measure the relative concentration of these end products on the two sides of the cytoplasmic membrane of anaerobically glycolyzing cells. Comparison of measured concentration rations to those expected at equilibrium for various transport modes indicates that acetate is a classical uncoupling agent, permeating the membrane oat comparable rates in the dissociated and undissociated forms. The lactate concentration ratio changes market markedly after an initial period of sustained glycolysis. This change is most readily explained as resulting from a lactate transport system that responds to an indicator of glycolytic activity. The data further indicates that lactate permeates the membrane in both dissociated and undissociated forms. Both acids, then are capable of catalytically dissipating the proton‐motives force. © 1995 John Wiley & Sons, Inc.
Effect of membrane morphology and operation on protein deposition in ultrafiltration membranes
Authors: Chen V. , Kim K. J. , Fane A. G.
Published: December 2004

Membrane morphology is compared to protein depostion under passive adsorption and ultrafiltration conditions. Solute resistance of protein deposits for membranes of varying roughness, structure, and permeability can vary dramatically with operating conditions. Using Brunauer–Emmett–Teller adsorption isotherm (BET), study of the internal area and accessibility of several uttrafiltration membranes to protein deposition allows better understanding of the fouling mechanisms and interpretation of adsorbed protein quantities. © 1995 John Wiley & Sons, Inc.
Effect of insulin on a serum‐free hybridoma culture
Authors: Zhou Weichang , Hu Wei‐Shou
Published: December 2004

Insulin is often included in serum‐free media for animal cell cultivation. However, the necessity of insulin for a specific cell line is rather uncertain. In this article we report the effects of insulin on the cultivation of a hybridoma cell line in a serum‐free medium. It was found that insulin affected neither the cell growth nor the antibody production. The specific growth rate and specific antibody production rate were very similar in the cultures with or without insulin. However, the presence of insulin affected the nutrient consumption rate and cell metabolism. Including insulin in the medium resulted in a higher specific glucose consumption rate, a shorter exponential growth stage, and a lower final antibody concentration. The elimination of insulin from the medium allowed antibody to accumulate to a concentration substantially higher than that in the insulin‐containing cultuvre. © 1995 John Wiley & Sons, Inc.
Photosynthetic performance of a helical tubular photobioreactor incorporating the cyanobacterium spirulina platensis
Authors: Watanabe Yoshitomo , de la Noüe Joël , Hall David O.
Published: December 2004

The photosynthetic performance of a helical tubular photobioreactor (“Biocoil”), incorporating the filamentous cyanobacterium Spirulina platensis, was investigated. The photobioreactor was constructed in a cylindrical shape (0.9 m high) with a 0.25‐m2basal area and a photostage comprising 60 m of transparent PVC tubing of 1.6‐cm inner diameter (volume = 12.1 L). The inner surface of the cylinder (area = 1.32 m2) was illuminated with cool white fluorescent lamps; the energy input of photosynthetically active radiation(PAR, 400 to 700 nm) into the photobioreactor was 2920 kJ per day. An air‐lift system ncorporating 4%CO2 was used to circulate the growth medium in the tubing. The maximum productivity achieved in batch culture was 7.18 g dry biomass per day [0.51 g · d biomass/L · day, or 5.44 g · d biomass/m2(inner surface of cylindrical shape)/day] which corresponded to a photosynthetic (PAR) efficiency of 5.45%. The CO2 was efficiently removed from the gaseous stream; monitoring the CO2 the outlet and inlet gas streams showed a 70% removal of CO2 from the inlet gas over an 8‐h period with almost maximum growth rate. © 1995 John Wiley & Sons, Inc.
Crossflow filtration of baker's yeast with periodical stopping of permeation flow and bubbling
Authors: Tanaka Takaaki , Itoh Hiroshi , Itoh Kazutaka , Nakanishi Kazuhiro , Kume Takaaki , Matsuno Ryuichi
Published: December 2004

The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 × 10−5 m3 /m2 s (290 L/m2 h) from 2 × 10−5 m3/m2 s (72 L/m2 h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux.© John Wiley & Sons, Inc.
Effects of p‐cresol and chlorophenols on pentachlorophenol biodegradation
Authors: Gu Yongxiang , Korus Roger A.
Published: December 2004

The effects of three aromatic compounds, p‐cresol, 2,4‐dichlorophenol (DCP), and 2,4,6‐trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p‐cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p‐cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. © 1995 John Wiley & Sons Inc.
Determination of effective diffusion coefficients and distribution constants in polysaccharide gels with non‐steady‐state measurements
Authors: Øyaas Jorun , Storrø Ivar , Lysberg Magne , Svendsen Hallvard , Levine David W.
Published: December 2004

A non‐steady‐state method has been used for determining the effective diffusion coefficient, De, and a distribution constant, Ki, of small molecules in alginate gel beads. A mathematical model based on Pick's law and includingexternal film diffusion resistance describe the diffusion process. Criticalexperimental parameters for the estimation of De and Ki, for both one‐ and two‐parameter methods were the initial solute concentration in the bulk liquid, the void fraction inthe reactor, and the experimental starting point. In our analysis, the two‐parameter method is preferable. Incorporation of an estimate of the film resistance into the overall model increased the estimated values of De significantly and improved the stability of the term over a range of reactor agitation rates. © 1995 John Wiley & Sons Inc.
Modeling of continuous Ph‐stat stirred tank reactor with Lactococcus lactis ssp. lactis bv. diacetylactis immobilized in calcium alginate gel beads
Authors: Cachon Rémy , Molin Paul , Diviès Charles
Published: December 2004

A dynamic diffusion–reaction–growth model is proposed for the study of lactic fermentation, the bioconversion of citric acid, and cell release in an immobilized cell reactor [pH‐stat continuous stirred tank‐reactor (CSTR)]. The model correctly simulates the onset of fermentation and colonization of the gel, followed by the steady state. External diffusion is nonlimiting and internal diffusion is limited by high cell densities at the periphery of the gel beads. Lactose–citrate cometabolism in the gel is related to the distribution of active included biomass within the gel and to gradients of substrates (lactose, citrate) and products (lactate, pH) in the beads. The utilization of lactose is limited by reaction, whereas that of citrate is limited by diffusion. Cell release from gel to the liquid medium occurs in the external spherical cap of the beads. In this peripheral zone viability is maintained at around 90%. © 1995 John Wiley & Sons Inc.
Importance of spore mutants for fed‐batch and continuous fermentation of Bacillus subtilis
Authors: Oh M. K. , Kim B. G. , Park S. H.
Published: December 2004

To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch‐type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed‐batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed‐batch culture as well as enable us to use very low dilution rates (<0.1 h−1) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h−1 the spollG mutant of Bacillus subtilis DB104 (Δnpr Δapr) (Emr) spollG (Bimr):: pMK101 (Cmr) showed a subtilisin yield about ten times higher than that from wild‐type DB104 (Δnpr Δapr)::pMK101 (Cmr). © 1995 John Wiley & Sons, Inc.
Enzyme‐mediated proteolysis of fibrous biopolymers: Dissolution front movement in fibrin or collagen under conditions of diffusive or convective transport
Authors: Anand Sriram , Wu Jung‐He , Diamond Scott L.
Published: December 2004

A numerical model based on the convective‐diffusive transport of reacting and adsorbing proteolytic enzymes within erodible fibrous biopolymers was used to predict lysis fronts moving across biogels such as fibrin or collagen. The fiber structure and the transport properties of solutes in fibrin (or collagen) were related to the local extent of dissolution within the dissolving structure. An accounting for solubilization of adsorbed species into solution from the eroding fiber phase provided for complete conservation of mass in reacting systems containing over 10 species. At conditions of fibrinolysis typical of clinical situations, the model accurately predicted the dynamic rate of lysis front movement for plasmin, urokinase, and tissue plasminogen activator (tPA)‐mediated lysis of fibrin gels measured in vitro. However, under conditions of extremely fast fibrinolysis using high enzyme concentrations, fibrinolytic fronts moved very rapidly (>0.1 mm/mm)—faster than predicted for diffusionlimited reactions—at nearly constant velocity for over 2 h, indicating non‐Fickian behavior. This was due to proteolysis‐mediated retraction of dissolving fibrin fibers that resulted in fiber convection and front‐sharpening within 3 μm of the reaction front, as observed by digitally enhanced microscopy. In comparing the model to fibrinolysis measurements using human lys77‐plasmin, the average first order rate constant for non‐crosslinked fibrin bond cleavage by fibrin‐bound plasmin was calculated to be 5s−1 assuming that 10 cleavages per fibrin monomer were required to solubilize each monomer. The model accurately predicted lysis front movement using pressure‐driven permeation of plasmin or urokinase into fibrin as well as literature data obtained under well‐ mixed conditions for tPA‐mediated fibrinolysis. This numerical formulation provides predictive capability for optimization of proteolytic systems which include thrombolytic therapy, wound healing, controlled drug release, and tissue engineering applications. © 1995 John Wiley & Sons, Inc.
Immobilization and properties of carminomycin 4‐O‐methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius
Authors: Scotti Claudio , Hutchinson C. Richard
Published: December 2004

The carminomycin 4‐O‐methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI‐activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S‐adenosyl‐L‐methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4°C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent Km (K′m) for carminomycin while the K′m for S‐adenosyl‐L‐methionine was approximately doubled. A 57% decrease in the Vmax value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. © 1995 John Wiley & Sons, Inc.
Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH
Authors: del Río Gabriel , Rodríguez Maria‐Elena , Munguía Maria‐Elena , LóPez‐Munguí Agustín , Soberón Xavier
Published: December 2004

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6‐aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide‐directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431‐Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half‐life of the mutant enzyme when stored at pH 8.5 as compared with the wild‐type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH‐stability profile. © 1995 John Wiley & Sons, Inc.