Showing papers in "Biotechnology and Bioengineering in 2001"

Dynamical model development and parameter identification for an anaerobic wastewater treatment process

Abstract: This paper deals with the development and the parameter identification of an anaerobic digestion process model. A two-step (acidogenesis-methanization) mass-balance model has been considered. The model incorporates electrochemical equilibria in order to include the alkalinity, which has to play a central role in the related monitoring and control strategy of a treatment plant. The identification is based on a set of dynamical experiments designed to cover a wide spectrum of operating conditions that are likely to take place in the practical operation of the plant. A step by step identification procedure to estimate the model parameters is presented. The results of 70 days of experiments in a 1-m(3) fermenter are then used to validate the model. (C) 2001 John Wiley & Sons, Inc.

Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FCγRIII

Abstract: The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.

Two-dimensional model of biofilm detachment caused by internal stress from liquid flow.

Abstract: A two-dimensional model for biofilm growth and detachment was used to evaluate the effect of detachment on biofilm structures. The detachment process is considered to be due to internal stress created by moving liquid past the biofilm. This model generated a variety of realistic biofilm-formation patterns. It was possible to model in a unified way two different biofilm detachment processes, erosion (small-particle loss), and sloughing (large-biomass-particle removal). The distribution of the fraction from total biomass detached as a function of detached particle mass, gives indications about which of the two mechanisms is dominant. Model simulations indicate that erosion makes the biofilm surface smoother. Sloughing, in contrast, leads to an increased biofilm-surface roughness. Faster growing biofilms have a faster detachment rate than slow-growing biofilms, under similar hydrodynamic conditions and biofilm strength. This is in perfect accordance with the experimental evidence showing that detachment is dependent on both shear- and microbial-growth rates. High growth rates trigger instability in biofilm accumulation and abrupt biomass loss (sloughing). Massive sloughing can be avoided by high liquid shear, combined with low biomass growth rates. As the modeling results show, the causes for sloughing must be sought not only in the biofilm strength, but also in its shape. Several "mushroom-like" biofilm structures like those repeatedly reported in the literature occurred, due to a combined effect of nutrient depletion and breaking at the colony base. A rough carrier surface promotes biofilm development in hydrodynamic conditions in which the biofilm on a flat surface would not form. Although biofilm patches filled completely the cavity in which they started to grow, they were unable to spill over the carrier peaks and to fully colonize the substratum.

Preparation, stability, and in vitro performance of vesicles made with diblock copolymers

Abstract: Vesicles made completely from diblock copoly- mers—polymersomes—can be stably prepared by a wide range of techniques common to liposomes. Pro- cesses such as film rehydration, sonication, and ex- trusion can generate many-micron giants as well as monodisperse, ~100 nm vesicles of PEO-PEE (polyethyl- eneoxide-polyethylethylene) or PEO-PBD (polyethyl- eneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as lipo- somes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal tran- sitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an ex- tended time with an excess of polymersomes. The ef- fects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micell- ization. The results potentiate a broad new class of tech- nologically useful, polymer-based vesicles. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 135-145, 2001.

Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor

Abstract: The goal of this study was to investigate the viability and synthetic function of rat hepatocytes cocultured with 3T3-J2 fibroblasts in a small-scale microchannel flat-plate bioreactor with and without an internal membrane oxygenator under flow. Bioreactor channel heights ranged between 85 and 500 microm and medium flow rates ranged between 0.06 and 4.18 mL/min. The results showed that the bioreactor without the oxygenator resulted in significantly decreased viability and function of hepatocytes, whereas hepatocytes in the bioreactor with internal membrane oxygenator were able to maintain their viability and function. The shear stress calculations showed that, at lower wall shear stresses (0.01 to 0.33 dyn/cm(2)), hepatocyte functions, measured as albumin and urea synthesis rates, were as much as 2.6- and 1.9-fold greater, respectively, than those at higher wall shear stresses (5 to 21 dyn/cm(2)). Stable albumin and urea synthesis rates for 10 days of perfusion were also demonstrated in the bioreactor with internal membrane oxygenator. These results are relevant in the design of hepatocyte bioreactors and the eventual scaling-up to clinical devices.

Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production.

Abstract: Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidated. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (P(trc) and P(lac), respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P(BAD) on a medium-copy plasmid produced 1.4-2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.

Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source.

Abstract: Succinic acid was produced by fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. When cells were anaerobically cultured in a medium containing 6.5 g/L glycerol, a high succinic acid yield (133%) was obtained while avoiding the formation of by-product acetic acid. The gram ratio of succinic acid to acetic acid was 25.8:1, which is 6.5 times higher than that obtained using glucose (ca. 4:1) as a carbon source. Therefore, succinic acid can be produced with much less by-product formation by using glycerol as a carbon source, which will facilitate its purification. When glucose and glycerol were cofermented with the increasing ratio of glucose to glycerol, the gram ratio of succinic acid to acetic acid and succinic acid yield decreased, suggesting that glucose enhanced acetic acid formation irrespective of the presence of glycerol. Glycerol consumption by A. succiniciproducens required unidentified nutritional components present in yeast extract. By intermittently feeding yeast extract along with glycerol, a high succinic acid yield (160%) could be obtained while still avoiding acetic acid formation. This resulted in the highest ratio of succinic acid to acetic acid (31.7:1).

Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose.

Abstract: Sixteen batch experiments were performed to evaluate the stability, kinetics, and metabolic paths of heat-shocked digester (HSD) sludge that transforms microcrystalline cellulose into hydrogen. Highly reproducible kinetic and metabolic data confirmed that HSD sludge could stably convert microcrystalline cellulose to hydrogen and volatile fatty acids (VFA) and induce metabolic shift to produce alcohols. We concluded that clostridia predominated the hydrogen-producing bacteria in the HSD sludge. Throughout this study the hydrogen percentage in the headspace of the digesters was greater than 50% and no methanogenesis was observed. The results emphasize that hydrogen significantly inhibited the hydrogen-producing activity of sludge when initial microcrystalline cellulose concentrations exceeded 25.0 g/L. A further 25 batch experiments performed with full factorial design incorporating multivariate analysis suggested that the ability of the sludge to convert cellulose into hydrogen was influenced mainly by the ratio of initial cellulose concentration (So) to initial sludge density (Xo), but not by interaction between the variables. The hydrogen-producing activity depended highly on interaction of So x (So/Xo). Through response surface analysis it was found that a maximum hydrogen yield of 3.2 mmol/g cellulose occurred at So = 40 g/L and So/Xo = 8 g cellulose/g VSS. A high specific rate of 18 mmol/(g VSS-d) occurred at So = 28 g/L and So/Xo = 9 g cellulose/g VSS. These experimental results suggest that high hydrogen generation from cellulose was accompanied by low So/Xo.

Regeneration of adenosine triphosphate from glycolytic intermediates for cell‐free protein synthesis

Abstract: A new approach for adenosine triphosphate (ATP) regeneration in a cell-free protein synthesis system is described. We first show that pyruvate can be used as a secondary energy source to replace or supplement the conventional secondary energy source, phosphoenol pyruvate (PEP). We also report that glucose-6-phosphate, an earlier intermediate of the glycolytic pathway, can be used for ATP regeneration. These new methods provide more stable maintenance of ATP concentration during protein synthesis. Because pyruvate and glucose-6-phosphate are the first and last intermediates of the glycolytic pathway, respectively, the results also suggest the possibility of using any glycolytic intermediate, or even glucose, for ATP regeneration in a cell-free protein synthesis system. As a result, the methods described provide cell-free protein synthesis with greater flexibility and cost efficiency.

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Abstract: Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells

Abstract: Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: Stoichiometry, kinetics, and the effect of pH.

Abstract: A metabolic model for the stoichiometry of acetate uptake under anaerobic conditions by an enriched culture of glycogen accumulating organisms (GAOs) was developed and tested by experimental studies. Glycogen served as the source of both reducing power and energy to drive the process of acetate uptake. The amount of glycogen consumed and poly-β-hydroxyvalerate (PHV) accumulated in the cells increased with increasing pH, indicating that the energy requirements for acetate uptake increased with pH. The composition of the accumulated poly-β-hydroxyalkanoates (PHAs) was adequately predicted using the assumption that acetyl-CoA and propionyl-CoA condense randomly to produce PHA. In addition, the rate of acetate uptake was strongly affected by the pH. The rate decreased with increasing pH and this dependence could be described with a saturation type of expression. A comparison of the rate of acetate uptake at low pH with the rates observed in enriched cultures of phosphorus accumulating organisms (PAOs) indicated that GAOs are able to compete effectively with PAOs in nutrient removal systems under certain conditions. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 17–31, 2001.

High‐cell‐density fermentation for production of L‐N‐carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter

Abstract: A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.

In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape.

Abstract: Successful closure of bone defects in patients remains an active area of basic and clinical research. A novel and promising approach is the transplantation of human bone marrow stromal cells (BMSCs), which have been shown to possess a significant osteogenic potential. The extent and quality of bone formation by transplanted human BMSCs strongly depends on the carrier matrix with which cells are transplanted; to date, hydroxyapatite/tricalcium phosphate (HA/TCP) supports far more osteogenesis than any other matrix tested. In order to further improve the technique of BMSC transplantation, we studied whether commercially available HA/TCP particles, clinically approved as an osteoconductive material and commercially available as particles measuring 0.5-1.0 mm diameter, is an optimum matrix for promoting bone development by BMSCs. HA/TCP and HA particles of varying size were sieved into a variety of size ranges, from <0.044 mm to 1.0-2.0 mm. Transplants were formed by mixing 40 mg aliquots of particles with cultured passaged human BMSCs. They were placed in subcutaneous pockets in immunocompromised Bg-Nu-XID mice and harvested 4 or 10 weeks later. The transplants were examined histologically; the presence of bone within each transplant was evaluated using histomorphometry or blindly scored on a semiquantitative scale. Transplant morphology and the amount of new bone varied in a consistent fashion based on particle size and shape. Transplants incorporating HA/TCP particles of 0.1-0.25 mm size demonstrated the greatest bone formation at both 4 and 10 weeks; larger or smaller particles were associated with less extensive bone formation, while a size of 0.044 mm represented a threshold below which no bone formation could be observed. Flat-sided HA particles measuring 0.1-0.25 mm formed no bone. The differences in bone formation were not attributable to the differences in cell attachment among the groups. Instead, the size and spatial and structural organization of the particles within BMSC transplants appear to determine the extent of bone formation. These findings provide necessary information for the successful clinical application of BMSC transplantation techniques.

Horseradish peroxidase catalyzed degradation of industrially important dyes.

Abstract: Horseradish peroxidase (HRP) is known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols. Here, for the first time we have shown HRP to be effective in degrading and precipitating industrially important azo dyes. For Remazol blue, the enzyme activity was found to be far better at pH 2.5 than at neutral pH. In addition, Remazol blue acts as a strong competitive inhibitor of HRP at neutral pH. Horseradish peroxidase shows broad substrate specificity toward a variety of azo dyes. Kinetic constants (K(m)(app) and V(max)(app)) for two different dyes have been determined. In addition to providing a systematic analysis of the potential of HRP in degradation of dyes, this study opens up a new area on exploration of commercial dyes as inhibitors of enzymes. 2001 John Wiley & Sons, Inc.

Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells

Abstract: Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.

Low-cost microbioreactor for high-throughput bioprocessing.

Abstract: The design of a microbioreactor is described. An optical sensing system was used for continuous measurements of pH, dissolved oxygen, and optical density in a 2 mL working volume. The K(L)a of the microbioreactor was evaluated under different conditions. An Escherichia coli fermentation in both the microbioreactor and a standard 1 L bioreactor showed similar pH, dissolved oxygen, and optical density profiles.%The low cost of the microbioreactor, detection system, and the small volume of the fermentation broth provide a basis for development of a multiple-bioreactor system for high-throughput bioprocess optimization.

α‐Chymotrypsin catalysis in imidazolium‐based ionic liquids

Abstract: The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by α-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF6]), and in combination with supercritical carbon dioxide (SC-CO2). The activity of α-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. α-Chymotrypsin freeze-dried with K2HPO4, KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF6] or [omim][PF6] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF6] than [bmim][PF6]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO2. These results indicate that [bmim][PF6] and [omim][PF6] provide a relatively polar environment, which can be modified with nonpolar SC-CO2 to optimize enzyme activity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 181–186, 2001.

Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae.

Abstract: An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.

Statistical reconciliation of the elemental and molecular biomass composition of Saccharomyces cerevisiae.

Abstract: A systematic mathematical procedure capable of detecting the presence of a gross error in the measurements and of reconciling connected data sets by using the maximum likelihood principle is applied to the biomass composition data of yeast. The biomass composition of Saccharomyces cerevisiae grown in a chemostat under glucose limitation was analyzed for its elemental and for its molecular composition. Both descriptions initially resulted in conflicting results concerning the elemental composition, molecular weight, and degrees of reduction. The application of the statistical reconciliation method, based on elemental balances and equality relations, is used to obtain a consistent biomass composition. Simultaneously, the error margins of the data sets are significantly reduced in the reconciliation process. On the basis of statistical analysis it was found that inclusion of about 4% water in the list of biomass constituents is essential to adequately describe the dry biomass and match both set of measurements. The reconciled carbon content of the biomass varied 4% from the ones obtained from the molecular analysis. The proposed method increases the accuracy of biomass composition data of its elements and its molecules by providing a best estimate based on all available data and thus provides an improved and consistent basis for metabolic flux analysis as well as black box modeling approaches.

N-removal in a granular sludge sequencing batch airlift reactor

Abstract: The removal of N-compounds in the sequencing batch airlift reactor (SBAR) containing granular sludge was studied under conditions of decreased dissolved oxygen (DO). A simulation model was developed to describe and evaluate the effects of several process conditions in the SBAR on N-removal performance. The model described the experimental data reasonable well. It has been shown that nitrification, denitrification, and removal of chemical oxygen demand (COD) can occur simultaneously in a granular sludge SBR. It has also been shown that the exact location of the autotrophic biomass influences the net N-removal. The distribution of the autotrophic biomass is influenced by the DO in the reactor. The optimal DO value is expected to be around 40% air saturation. It was shown that storage and subsequent degradation of poly-beta-hydroxybutyrate (PHB) benefit the denitrification. In particular, PHB is stored in bacteria situated in deeper layers of the granules below where the autotrophic activity occurs, serves as a C-source for denitrification.

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis.

Abstract: A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, α-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound α-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized α-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass–enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons Biotechnol Bioeng 74: 249–255, 2001.

Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors.

Abstract: Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%). Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity. Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity. Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS). Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye. Hydrogen, added in bulk, was clearly the preferred electron donor. Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge. However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction. The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye. The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria.

Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis.

Abstract: Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.

Effects of mixing intensity on tissue-engineered cartilage.

Abstract: Mechanical forces regulate the structure and function of many tissues in vivo; recent results indicate that the mechanical environment can decisively influence the development of engineered tissues cultured in vitro. To investigate the effects of the hydrodynamic environment on tissue-engineered cartilage, primary bovine calf chondrocytes were seeded on fibrous polyglycolic acid meshes and cultured in spinner flasks either statically or at one of nine different turbulent mixing intensities. In medium from unmixed flasks, CO(2) accumulated and O(2) was depleted, whereas in medium from mixed flasks the concentrations of both gases approached their equilibrium values. Relative to constructs exposed to nonmixed conditions, constructs exposed to mixing contained higher fractions of collagen, synthesized and released more GAG, but contained lower fractions of GAG. Across the wide range of mixing intensities investigated, the presence or absence of mixing, but not the intensity of the mixing, was the primary determinant of the GAG and collagen content in the constructs. The all-or-none nature of these responses may provide insight into the mechanism(s) by which engineered cartilage perceives changes in its hydrodynamic environment and responds by modifying extracellular matrix production and release. 2001 John Wiley & Sons, Inc.

Engineering of a mammalian cell line for reduction of Lactate formation and high monoclonal antibody production

Abstract: Lactate and ammonia are the two major waste products formed during mammalian cell growth. Accumulation of these side products can have a negative effect on cell growth, and has drawn recent attention because of their inhibitory effects on the specific product synthesis rate. Our aim is to reduce lactate formation in the cell culture by genetically manipulating of the pathway of lactate synthesis with an aim to achieve high monoclonal antibody production. We have partially disrupted the LDH-A gene by homologous recombination in hybridoma cells (ATCC-CRL-1606). The cells that received the newly introduced DNA were selected by G418, and an LDH-deficient cell was identified by a screening method based on medium color changing in 96-well plates. A variant cell, LDH-neo21, was identified through this screening method and was characterized. The specific productivity of lactate by LDH-neo21 cells was 50% lower than that of parental cells. Intracellular LDH enzyme activity was significantly reduced. The cell growth was improved both in terms of cell density and cell viability. Total cell density potentially reached 5 x 10(6) cells/mL while the parental hybridoma cells had a cell density of 3.5 x 10(6) cells/mL, which represented a 30% increase. The antibody production of LDH-neo21 cells was threefold greater than that of parental cells during 5-day batch culture. Polymerase chain reaction (PCR) results showed that at least one copy of the LDH-A gene was disrupted in the LDH-neo21 cells. The variant of the hybridoma cell exhibited a significant advantage of reduced lactate formation in the cell culture with a high concentration of glucose, which led to a higher production of monoclonal antibody. 2001 John Wiley & Sons, Inc.

Process monitoring of an industrial fed-batch fermentation.

Abstract: Market demand places great emphasis in industry on product quality. Consequently, process monitoring and control have become important aspects of systems engineering. In this article we detail the results of a 2-year study focusing on the development of a condition monitoring system for a fed-batch fermentation system operated by Biochemie Gmbh in Austria. We also demonstrate the suitability and limitations of current state of the art technologies in this field and suggest novel modifications and configurations to improve their suitability for application to a fed-batch fermentation system.

Characterization of monoclonal antibody fragments produced by plant cells.

Abstract: Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120- and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.

Probing the Performance Limits of the Escherichia coli Metabolic Network Subject to Gene Additions or Deletions

Abstract: An optimization-based procedure for studying the response of metabolic networks after gene knockouts or additions is introduced and applied to a linear flux balance analysis (FBA) Escherichia coli model. Both the gene addition problem of optimally selecting which for- eign genes to recombine into E. coli, as well as the gene deletion problem of removing a given number of existing ones, are formulated as mixed-integer optimization problems using binary 0-1 variables. The developed modeling and optimization framework is tested by inves- tigating the effect of gene deletions on biomass produc- tion and addressing the maximum theoretical production of the 20 amino acids for aerobic growth on glucose and acetate substrates. In the gene deletion study, the small- est gene set necessary to achieve maximum biomass production in E. coli is determined for aerobic growth on glucose. The subsequent gene knockout analysis indi- cates that biomass production decreases monotonically, rendering the metabolic network incapable of growth af- ter only 18 gene deletions. In the gene addition study, the E. coli flux balance model is augmented with 3,400 non- E. coli reactions from the KEGG database to form a mul- tispecies model. This model is referred to as the Univer- sal model. This study reveals that the maximum theoret- ical production of six amino acids could be improved by the addition of only one or two genes to the native amino acid production pathway of E. coli, even though the model could choose from 3,400 foreign reaction candi- dates. Specifically, manipulation of the arginine produc- tion pathway showed the most promise with 8.75% and 9.05% predicted increases with the addition of genes for growth on glucose and acetate, respectively. The mecha- nism of all suggested enhancements is either by: 1) im- proving the energy efficiency and/or 2) increasing the carbon conversion efficiency of the production route. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 364-375, 2001.

Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs.

Abstract: Microbial control of biogenic production of hydrogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cultures enriched from produced water of a Canadian oil reservoir. The presence of nitrate at concentrations up to 20 mM had little effect on the rate of sulfate reduction by a pure culture of Lac6. Addition of CVO imposed a strong inhibition effect on production of sulfide. In the absence of added nitrate SRB we were able to overcome this effect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H2S immediately. The concentration of sulfide decreased to a negligible level due to nitrate-dependent sulfide oxidation activity of CVO. This was not prevented by raising the concentration of Na-lactate, the electron donor for sulfate reduction. Similar results were obtained with enrichment cultures. Enrichments of produced water with sulfide and nitrate were dominated by CVO, whereas enrichments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB enrichment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concentration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pronounced lag time, it was needed for emergence of a sizable CVO population. The results of the present study show that two mechanisms are involved in microbial control of biogenic sulfide production. First, addition of NR-SOB imposes an inhibition effect, possibly by increasing the environmental redox potential to levels which are inhibitory for SRB. Second, in the presence of sufficient nitrate, NR-SOB oxidize sulfide, leading to its complete removal from the environment. Successful microbial control of H2S in an oil reservoir is crucially dependent on the simultaneous presence of NR-SOB (either indigenous population or injected) and nitrate in the environment.