bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of mixing rate on β‐carotene production and extraction by dunaliella salina in two‐phase bioreactors
Authors: Hejazi M. A. , Andrysiewicz E. , Tramper J. , Wijffels R. H. 
Published: October 2003

Abstractβ‐Carotene has many applications in the food, cosmetic, and pharmaceutical industries; Dunaliella salina is currently the main source for natural β‐carotene. We have investigated the effect of mixing rate and whether it leads to the facilitated release of β‐carotene from the cells of Dunaliella salina in two‐phase bioreactors. Three pairs of bioreactors were inoculated at the same time, operated at 100, 150, and 170 rounds per minute, respectively, and illuminated with a light intensity of 700 μmol m−2 s−1. Each pair consisted of one bioreactor containing only aqueous phase for the blank and one containing the water phase together with dodecane, which is biocompatible with the cells. Comparison of the viability and growth of the cells grown under different agitation rates shows that 170 rpm and 150 rpm are just as good as 100 rpm. The presence and absence of the organic phase also has no influence on the viability and growth of the cells. In contrast to the growth rate, the extraction rate of β‐carotene is influenced by the stirrer speed. The extraction rate increases at a higher stirring rate. The effectiveness of extraction with respect to power input is comparable for all the applied mixing rates, even though it is slightly lower for 100 rpm than the others. The chlorophyll concentration in the organic phase remained very low during the experiment, although at higher mixing rates, chlorophyll impurity increased up to 3% (w/w) of the total extracted pigments. At 170 rpm carotenoid and chlorophyll undergo the highest extraction rate for both pigments—0.5% of the chlorophyll and 6% of the carotenoid is extracted. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 591–596, 2003.
Higher expression of fab antibody fragments in a CHO cell line at reduced temperature
Authors: Schatz Simone M. , Kerschbaumer Randolf J. , Gerstenbauer Gabriele , Kral Martina , Dorner Friedrich , Scheiflinger Friedrich 
Published: October 2003

AbstractA chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two‐stage production process. Cells were first grown to 90% confluence at 37°C in a proliferation phase, followed by a production phase at either 37°C or 28°C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28°C resulted in a 14‐fold increase in product yield when cells were cultivated in serum‐containing medium, and in a 38‐fold increase in product yield when serum‐free medium was applied. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 433–438, 2003.
Optical processing of bacterial libraries for directed evolution
Authors: Scruggs Allan Wallace , Woodbury Neal W. 
Published: October 2003

AbstractSelection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer‐controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56‐fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 445–451, 2003.
Development of a fed‐batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat
Authors: Chao Yun‐Peng , Chern Jong‐Tzer , Lin Wei Shing , Wang Zei Wen 
Published: October 2003

AbstractTo effectively achieve tight regulation and high‐level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30°→41°C stepwise upshift, the shake‐flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30°C. By exploiting the fed‐batch fermentation approach, the recombinant cell batch initially kept at 30°C in a lab‐scale fermentor was exposed to 41°C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37°C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880‐fold over that obtained at the shake‐flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale‐up of recombinant proteins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 459–466, 2003.
Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in escherichia coli
Authors: Seo Jeong Hyun , Li Lin , Yeo Joo Sang , Cha Hyung Joon 
Published: October 2003

AbstractBaculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein (∼58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin–GFP fusion protein showed higher cell growth (∼1.8‐fold) and higher GFP yield (∼3.5‐fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by α‐chymotrypsin, a protease that has similar properties to insect midgut polyhedra‐associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 467–473, 2003.
Supplementation‐dependent differences in the rates of embryonic stem cell self‐renewal, differentiation, and apoptosis
Authors: Viswanathan Sowmya , Benatar Tania , Mileikovsky Maria , Lauffenburger Douglas A. , Nagy Andras , Zandstra Peter W. 
Published: October 2003

AbstractAlthough it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4‐GFP‐positive (ES) and ‐negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose‐dependent survival and proliferation advantage, thus enabling the long‐term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor‐4 (FGF‐4), or insulin‐like growth factor‐1 (IGF‐1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 505–517, 2003.
Degradation of xenobiotics in a partitioning bioreactor in which the partitioning phase is a polymer
Authors: Amsden Brian G. , Bochanysz Justyna , Daugulis Andrew J. 
Published: October 2003

AbstractTwo‐phase partitioning bioreactors (TPPBs) are characterized by a cell‐containing aqueous phase and a second immiscible phase that contains toxic and/or hydrophobic substrates that partition to the cells at subinhibitory levels in response to the metabolic demand of the organisms. To date, the delivery phase in TPPBs has been a hydrophobic solvent that traditionally needed to possess a variety of important properties including biocompatibility, nonbioavailability, low volatility, and low cost, among others. In the present work we have shown that the organic solvent phase can be replaced by inexpensive polymer beads that function in a similar fashion as organic solvents, delivering a toxic substrate to cells based on equilibrium considerations. Specifically, 3.4 mm diameter beads of poly(ethylene‐co‐vinyl acetate) (EVA) were used to reduce the aqueous concentration of phenol in a bioreactor from toxic levels ( ∼2,000 mg/L) to subinhibitory levels (∼750 mg/L), after which Pseudomonas putida ATCC 11172 was added to the system and allowed to consume the total phenol loading. Thus, the beads absorbed the toxic substrate and released it to the cells on demand. The EVA beads, which could be reused, were able to absorb 14 mg phenol/g EVA. This work has opened the possibility of using widely mixed cultures in TPPB systems without concern for degradation of the delivery material and without concern of contamination. © 2003 Wiley Periodicals. Biotechnol Bioeng 84: 399–305, 2003.
Further studies related to the scale‐up of high cell density escherichia coli fed‐batch fermentations:
Authors: Onyeaka Helen , Nienow Alvin W. , Hewitt Christopher J. 
Published: October 2003

AbstractIn this work, we report on the further development of the scale‐down, two‐compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli fed‐batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large‐scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m3 industrial fed‐batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well‐mixed, 5L laboratory scale case. However, by using a combination of the well‐mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m3 reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO2 and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7. © 2003 Wiley Periodicals. Biotechnol Bioeng 84: 474–484, 2003.
Catalytic features of monoclonal antibody i41SL1‐2 subunits
Authors: Hifumi Emi , Kondo Hiroyuki , Mitsuda Yukie , Uda Taizo 
Published: October 2003

AbstractA monoclonal antibody (mAb), i41SL1‐2, was obtained by immunizing the peptide of complementarity‐determining region‐1 (CDRL‐1: RSSKSLLYSNGNTYLY) of a super catalytic antibody light chain, 41S‐2‐L, capable of enzymatically destroying the gp41 molecule of the HIV‐1 envelope. From the DNA and the deduced amino acid sequences of the light and heavy chain of i41SL1‐2 mAb, molecular modeling was conducted that suggested that both subunits of i41SL1‐2 mAb possess catalytic triads in their structures. Especially the light chain of i41SL1‐2 mAb possesses a characteristic catalytic triad composed of Asp1, Ser27A, and His93, whose positions are identical to the catalytic antibody light chain, VIPase, of S. Paul and colleagues (see text). The antibody gene of i41SL1‐2 light chain and VIPase belong to the same germline, bd2, suggesting that the discrete germline inherently possesses catalytic activity. Both light and heavy chains of i41SL1‐2 mAb degraded the antigenic peptide CDRL‐1 within 47 and 57 h, respectively. The catalytic reaction constant (kcat) of the light and heavy chain was 6.1 × 10−1 and 6.2 × 10−1 min−1, respectively. These are high values for the natural catalytic antibodies reported so far. The catalytic efficiency (kcat/Km) of the light and heavy chain was 3.1 × 105 and 4.9 × 104 M−1 min−1, respectively. The first cleaved bond of the antigenic peptide by subunits of i41SL1‐2 mAb was between Arg1 and Ser2 in the sequence of CDRL‐1, suggesting a serine protease character. © 2003 Wiley Periodicals. Biotechnol Bioeng 84: 485–493, 2003.
Dynamic behavior of adsorber membranes for protein recovery
Authors: Avramescu Maria‐Elena , Borneman Zandrie , Wessling Matthias 
Published: October 2003

AbstractIn recent years there has been a considerable interest in developing membrane chromatography systems that function as a short, wide chromatographic column in which the adsorptive packing consists of one or more microporous membranes. This study reports the use of new adsorber membranes prepared by the incorporation of various types of ion exchange resins into an EVAL porous membrane for protein recovery. The obtained heterogeneous matrixes composed of solid particles surrounded by the polymeric film possess a good accessibility for the protein to the adsorptive sites. Furthermore, small particles can be embedded into porous polymeric structures without the disadvantages of classical chromatographic columns (high pressure drop, fouling and plugging sensitivity, low flow rate), but with the advantages of membrane technology (easy scale‐up, low‐pressure drop, high flow rate). The adsorptive membranes feature high static as well as dynamic protein adsorption capacities for operating flow rates ranging from 200 to 400 L h bar per m2 and ionic strength of 20–200 mM. In a sequential desorption step by changing the pH and/or the ionic strength of the eluent, up to 90% protein recovery was obtained. Next to the separation, the mixed matrix adsorber membrane functions as a concentration medium since the protein can be concentrated up to 20‐fold in the eluent. The adsorber membranes can be reused in multiple adsorption/desorption cycles with good adsorption performances. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 564–572, 2003.
On‐line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy
Authors: Cannizzaro Christopher , Gügerli Raphael , Marison Ian , von Stockar Urs 
Published: October 2003

AbstractIn this work, dielectric spectroscopy was used to monitor two CHO perfusion culture experiments (B14 and B16). The capacitance of the cell suspension was recorded every 20 minutes over an excitation frequency range of 0.2 MHz to 10.0 MHz. A phase plot of the capacitance at a low excitation frequency vs. the value at a higher frequency proved to be an accurate indicator of the major transition points of the culture, i.e., maximum cell viability, end of lactate consumption, point of zero viability. For both experiments, the capacitance signal correlated very well (R2 >0.98) with viable cell number up to concentrations of 1 × 107 cells/mL. Visual observation of the capacitance spectra indicated that changes in the capacitance relative to frequency were related to the cellular morphology. A multivariate model was developed using off‐line data that could predict the median cell diameter within a single experiment (B14) with an error of 0.34 μm (2%). Upon extension to a subsequent experiment (B16), the predicted error was 1.18 μm (9%). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 597–610, 2003.
Purification of histidine‐tagged single‐chain Fv‐antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers
Authors: Kumar Ashok , Wahlund Per‐Olof , Kepka Cecilia , Galaev Igor Yu , Mattiasson Bo 
Published: October 2003

AbstractMetal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well‐characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal‐loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N‐isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine‐tagged single‐chain Fv‐antibody fragments (His6‐scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein‐polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His6‐scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS‐PAGE pattern showed significant purification of the antibody fragments. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 494–503, 2003.
Primmorphs from archaeocytes‐dominant cell population of the sponge hymeniacidon perleve: improved cell proliferation and spiculogenesis
Authors: Zhang Xiaoying , Cao Xupeng , Zhang Wei , Yu Xingju , Jin Meifang 
Published: October 2003

AbstractMarine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge‐derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed‐cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte‐dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed‐cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time‐dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge‐derived drugs. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 583–590, 2003.
Bioscrubbing of waste gas—substrate absorber to avoid instability induced by inhibition kinetics
Authors: Oliveira Teresa A. C. , Livingston Andrew G. 
Published: October 2003

AbstractThe stability of a continuous stirred tank bioreactor treating a gas stream containing monochlorobenzene (MCB) was studied theoretically and experimentally. A bioreactor inoculated with Pseudomonas sp. strain JS150 was submitted to successive step disturbances in the MCB load, inducing washout and system instability. With time, and subject to high MCB concentrations in the biomedium, the microorganisms appeared to adapt to high MCB load, and needed increasingly severe shocks to induce washout. To improve the bioreactor stability, the influence of an MCB‐absorber prior to the bioreactor was investigated, using silicone oil as the absorbent for MCB. A parallel was established with the first set of experiments (no absorber). Phase plane plots showed how the presence of the absorber changed the system trajectories from washout into stable pseudo‐steady states. Experimental results confirmed the benefits of the absorber in avoiding washout under high MCB loads. At periods of low loading, MCB was desorbed from the absorber. For the same loading conditions, removal efficiencies were much higher than when no absorber was present. Elimination capacities observed in the bioreactor were much higher than those previously reported for biotrickling filters treating MCB containing gas streams: 300 to 450 g m−3 h−1. Gas inlet concentrations were in the range 12 to 65 g m−3, well above the 5 g m−3 upper limit usually suggested for biological treatment of waste gases, showing that highly concentrated gas streams may be biologically treated so long as inhibitory concentrations are not reached in the bioreactor. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 552–563, 2003.