Showing papers in "Biotechnology and Bioengineering in 1998"
TL;DR: These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the largeHydrophobic active centers of their "open and hyperactivated structure", which becomes a very promising immobilization method with general application for most lipases.
Abstract: A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.
510 citations
TL;DR: Quantitative two- and three-dimensional models were evaluated by introducing statistical measures to characterize the complete biofilm structure, both the surface structure and volume structure, showing the change from a compact and dense biofilm to a highly porous and open biofilm.
Abstract: A hybrid differential-discrete mathematical model has been used to simulate biofilm structures (surface shape, roughness, porosity) as a result of microbial growth in different environmental conditions. In this study, quantitative two- and three-dimensional models were evaluated by introducing statistical measures to characterize the complete biofilm structure, both the surface structure and volume structure. The surface enlargement, coefficient of roughness, fractal dimension of surface, biofilm compactness, and solids hold-up were found to be good measures of biofilm structure complexity. Among many possible factors affecting the biofilm structure, the influence of biomass growth in relation to the diffusive substrate transport was investigated. Porous biofilms, with many channels and voids between the "finger-like" or "mushroom" outgrowth, were obtained in a substrate-transport-limited regime. Conversely, compact and dense biofilms occurred in systems limited by the biomass growth rate and not by the substrate transfer rate. The surface complexity measures (enlargement, roughness, fractal dimension) all increased with increased transport limitation, whereas the volume measures (compactness, solid hold-up) decreased, showing the change from a compact and dense to a highly porous and open biofilm.
499 citations
TL;DR: An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic and thermophilic conditions and large fluctuations were observed in methanogenic population levels as volatile fatty acids accumulated and were subsequently consumed.
Abstract: An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester.
392 citations
TL;DR: This work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase, which may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion.
Abstract: Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.
349 citations
TL;DR: It is proposed that largesolutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix.
Abstract: Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes (>5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix.
329 citations
TL;DR: The study showed that, when the effect of light saturation is eliminated or reduced, productivity and solar irradiance are linearly correlated even at very high diurnal irradiance values, and supported findings that outdoor algal cultures are light-limited even during bright summer days.
Abstract: The light saturation effect imposes a serious limitation on the efficiency with which solar energy can be utilized in outdoor algal cultures. One solution proposed to reduce the intensity of incident solar radiation and overcome the light saturation effect is "spatial dilution of light" (i.e., distribution of the impinging photon flux on a greater photosynthetic surface area), but consistent experimental data supporting a significant positive influence of spatial light dilution on the productivity and the photosynthetic efficiency of outdoor algal cultures have never been reported. We used a coiled tubular reactor and compared a near-horizontal straight tubular reactor and a near-horizontal flat panel in outdoor cultivation of the cyanobacterium Arthrospira (Spirulina) platensis under defined operating conditions for optimum productivity. The photosynthetic efficiency achieved in the tubular systems was significantly higher because their curved surface "diluted" the impinging solar radiation and thus reduced the light saturation effect. This interpretation was supported by the results of experiments carried out in the laboratory under continuous artificial illumination using both a flat and a curved chamber reactor. The study also showed that, when the effect of light saturation is eliminated or reduced, productivity and solar irradiance are linearly correlated even at very high diurnal irradiance values, and supported findings that outdoor algal cultures are light-limited even during bright summer days. It was also observed that, besides improving the photosynthetic efficiency of the culture, spatial dilution of light also leads to higher growth rates and lowers the cellular content of accessory pigments; that is, it reduces mutual shading in the culture. The inadequacy of using volumetric productivity as the sole criterion for comparing reactors of different surface-to-volume ratio and of the areal productivity for evaluating the performance of elevated photobioreactors operated outdoors is stressed; it is furthermore suggested that the photosynthetic efficiency achieved by the culture also be calculated to provide a suitable parameter for comparison of different algal cultivation systems operated under similar climatic conditions. Copyright 1998 John Wiley & Sons, Inc.
310 citations
TL;DR: The system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM, which may find clinical applications and provide a tool to study molecular mechanisms in vascular development.
Abstract: The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM compo- nent with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture condi- tions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 ◊ 5 mm, 2- mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer ma- trices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matri- ces was a function of cell concentration in the seeding solution, but under all conditions a larger number (ap- proximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were ob- tained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture condi- tions were next studied by culturing cell-polymer con- structs in a stirred bioreactor versus static culture condi- tions. The stirred culture of SMC-seeded polymer matri- ces resulted in tissues with a cell density of 6.4 ± 0.8 ◊ 10 8 cells/cm 3 after 5 weeks, compared to 2.0 ± 1.1 ◊ 10 8 cells/ cm 3 with static culture. The elastin and collagen synthe- sis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred biore- actor was 24 ± 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were im- planted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and pro- vide a tool to study molecular mechanisms in vascular development. © 1998 John Wiley & Sons, Inc. Biotechnol Bio- eng 57: 46-54, 1998.
300 citations
TL;DR: A sensitivity study of six key parameters in the interfacial photopolymerization process was performed to aid in determination of the optimal encapsulation conditions, leading to the most uniform hydrogel membranes and viable islets.
Abstract: A method has been defined to interfacially photopolymerize poly(ethylene glycol) diacrylates (PEG diacrylates) to form a crosslinked hydrogel membrane upon the surfaces of porcine islets of Langerhans to serve as an immune barrier for allo- and xenotransplantation. A sensitivity study of six key parameters in the interfacial photopolymerization process was performed to aid in determination of the optimal encapsulation conditions, leading to the most uniform hydrogel membranes and viable islets. The key parameters included the concentrations of the components of the initiation scheme, namely eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone. Other parameters investigated included the duration and flux of laser irradiation and the PEG diacrylate molecular weight. Each parameter was doubled and halved from the standard conditions used in the encapsulation process while holding all the remaining parameters at the standard conditions. The effects of changing each parameter on islet viability, encapsulation efficiency, and gel thickness were quantified. Islet viability was sensitive to the duration of laser illumination, viability significantly increasing as the duration was reduced. Encapsulation efficiency was sensitive to the concentrations of eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone, to the laser flux, and to the PEG diacrylate molecular weight. Increasing the concentration of eosin Y significantly improved the encapsulation efficiency, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the encapsulation efficiency. Gel thickness was sensitive to the concentrations of triethanolamine and 1-vinyl 2-pyrrolidinone, to the duration of laser illumination, and to the PEG diacrylate molecular weight. Increasing the PEG diacrylate molecular weight significantly increased the gel thickness, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the gel thickness. From this sensitivity study, conditions were determined to encapsulate porcine islets, resulting in greater than 90% islet viability and greater than 90% encapsulation efficiency.
272 citations
TL;DR: The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect, and it is hypothesized that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain.
Abstract: Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40 degrees C) and enzyme loading (0.16 micromol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 micromol/g at 4 degrees C and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40 degrees C when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4 degrees C, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain.
267 citations
TL;DR: It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector; Tween 20 is slightly more effective than Tween 80.
Abstract: Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. (The critical relationship was the Tween loading on the biomass, not the Tween concentration in solution.) The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 degrees C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. Copyright 1998 John Wiley & Sons, Inc.
254 citations
TL;DR: Following a high-cell-density fermentation which provided biomass up to 48 g cell dry weight per liter, alternating osmotic shocks in combination with crossflow filtration techniques to harvest the compatible solutes ectoine and hydroxyectoine were applied.
Abstract: A novel biotechnological process called "bacterial milking" has been established for the production of compatible solutes using the Gram-negative bacterium Halomonas elongata. Following a high-cell-density fermentation which provided biomass up to 48 g cell dry weight per liter, we applied alternating osmotic shocks in combination with crossflow filtration techniques to harvest the compatible solutes ectoine and hydroxyectoine. H. elongata, like other halophilic or halotolerant microorganisms, produces compatible solutes in response to the salinity of the medium. When transferred to a low salinity medium (osmotic downshock), H. elongata cells rapidly released their solutes to achieve osmotic equilibrium. Subsequent reincubation in a medium of higher salt concentration resulted in resynthesis of these compatible solutes and-after a defined regeneration time-the procedure could be repeated. By repeatedly performing this "bacterial milking" process (at least nine times) we were able to produce large amounts of ectoines with a biomass productivity of 155 mg of ectoine per cycle per gram cell dry weight. Further purification of the products was achieved by a simple two-step procedure based on cation exchange chromatography and crystallization. The principles described in this article may also be useful for the production of other low-molecular-weight compounds.
TL;DR: Observations confirmed a hypothesis that the balance between biofilm substrate surface loading and detachment force determines the biofilm structure, and that with the right balance, smooth, dense and stable biofilms can be obtained.
Abstract: The influence of process conditions (substrate loading rate and detachment force) on the structure of biofilms grown on basalt particles in a Biofilm Airlift Sus- pension (BAS) reactor was studied. The structure of the biofilms (density, surface shape, and thickness) and mi- crobial characteristics (biomass yield) were investigated at substrate loading rates of 5, 10, 15, and 20 kg COD/ m 3 z day with basalt concentrations of 60 g/L, 150 g/L, and 250 g/L. The basalt concentration determines the number of biofilm particles in steady state, which is the main determining factor for the biofilm detachment in these systems. In total, 12 experimental runs were per- formed. A high biofilm density (up to 67 g/L) and a high biomass concentration was observed at high detach- ment forces. The higher biomass content is associated with a lower biomass substrate loading rate and there- fore with a lower biomass yield (from 0.4 down to 0.12 gbiomass/gacetate). Contrary to general beliefs, the ob- served biomass detachment decreased with increasing detachment force. In addition, smoother (fewer protuber- ances), denser and thinner compact biofilms were ob- tained when the biomass surface production rate de- creased and/or the detachment force increased. These observations confirmed a hypothesis, postulated earlier by Van Loosdrecht et al. (1995b), that the balance be- tween biofilm substrate surface loading (proportional to biomass surface production rate, when biomass yield is constant) and detachment force determines the biofilm structure. When detachment forces are relatively high only a patchy biofilm will develop, whereas at low de- tachment forces, the biofilm becomes highly heteroge- neous with many pores and protuberances. With the right balance, smooth, dense and stable biofilms can be obtained. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 400-407, 1998.
TL;DR: Recombinant Chinese hamster ovary cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX).
Abstract: Previously, the highest producing (HP) recombinant CHO subclones isolated at various methotrexate (MTX) levels showed different antibody production stability during long-term culture, although they were clonally derived from CS13 transformant. In this study, genetic basis for their difference in antibody production stability was investigated using southern blot hybridization and fluorescence in situ hybridization (FISH) techniques. Southern analysis of HP subclones revealed that light-chain (LC) and heavy-chain (HC) cDNAs were located closely within 23 kb on an amplification unit, and the configuration of LC and HC cDNAs within this amplification unit was not disrupted during long-term culture in the absence of MTX. However, when LC and HC genes were localized on the metaphase chromosomes of HP subclones using FISH, the amplified sequences were present as an extended array on diverse marker chromosomes. HP subclones selected at higher MTX level had more kinds of marker chromosomes. CS13*-002 isolated at 0.02 microM MTX had only one marker chromosome (m002), whereas CS13*-1.0 isolated at 1 microM MTX had five different ones (m10A, m10B, m10C, m10D, and m10E). Each marker chromosome showed different fate during long-term culture of HP subclones in the absence of MTX, resulting in different degrees of stability among the HP subclones. The m10A and m10B remained unchanged, whereas the others disappeared or evolved to variants with shortened amplified arrays. The cells containing stable marker chromosomes constituted dominant subpopulations in CS13*-1.0, and thereby CS13*-1.0 became most stable in regard to antibody production during long-term culture. Furthermore, our dual-color FISH showed that the telomeric ends of amplified arrays on the stable marker chromosomes were always surrounded by (TTAGGG)(n) sequences, indicating that (TTAGGG)(n) sequences are closely related to the stability and evolution of amplified sequences. Taken together, our data show that the assessment of genotypic stability of amplified CHO cells is a prerequisite for understanding their production stability during long-term culture in the absence of selection pressure.
TL;DR: Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutionsof ammonium sulphate, sodium chloride, potassium isothiocyanate, and potassium chloride.
Abstract: Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.
TL;DR: The concept of balancing the elemental composition of growth medium with biomass composition to obtain high-density cultures is introduced and the performance of high cell density photobioreactors can be significantly enhanced by proper medium design.
Abstract: The basic requirements for high-density photoautotrophic microalgal cultures in enclosed photobioreactors are a powerful light source and proper distribution of light, efficient gas exchange, and suitable medium composition. This article introduces the concept of balancing the elemental composition of growth medium with biomass composition to obtain high-density cultures. N-8 medium, commonly used for culturing Chlorella vulgaris was evaluated for its capacity to support high-density cultures on the basis of elemental stoichiometric composition of C. vulgaris. This analysis showed that the N-8 medium is deficient in iron, magnesium, sulfur, and nitrogen at high cell densities. N-8 medium was redesigned to contain stoichiometrically balanced quantities of the four deficient elements to support a biomass concentration of 2% (v/v). The redesigned medium, called M-8 medium, resulted in up to three- to fivefold increase in total chlorophyll content per volume of culture as compared to N-8 medium. Further experiments showed that addition of each of the four elements separately to N-8 medium did not improve culture performance and that balanced supplementation of all four deficient elements was required to yield the improved performance. Long-term (24 d) C. vulgaris culture in M-8 medium showed continuous increase in chlorophyll content and biomass throughout the period of cultivation. In contrast, the increase in chlorophyll content and biomass ceased after 7 and 12 d, respectively in N-8 medium, demonstrating the higher capacity of M-8 medium to produce biomass. Thus, the performance of high cell density photobioreactors can be significantly enhanced by proper medium design. The elemental composition of the biomass generated is an appropriate basis for medium design.
TL;DR: A macromodel is developed for estimating the year-long biomass productivity of outdoor cultures of microalga in tubular photobioreactors, taking into account photoinhibition and photolimitation, and reproducing the experimental results with less than 20% error.
Abstract: A macromodel is developed for estimating the year-long biomass productivity of outdoor cultures of microalga in tubular photobioreactors. The model evaluates the solar irradiance on the culture surface as a function of day of the year and the geographic location. In a second step, the geometry of the system is taken into account in estimating the average irradiance to which the cells are exposed. Finally, the growth rate is estimated as a function of irradiance, taking into account photoinhibition and photolimitation. The model interconnects solar irradiance (an environmental variable), tube diameter (a design variable), and dilution rate (an operating variable). Continuous cultures in two different tubular photobioreactors were analyzed using the macromodel. The biomass productivity ranged from 0.50 to 2.04 g L−1 d−1, and from 1.08 to 2.76 g L−1 d−1, for the larger and the smaller tube diameter photobioreactors, respectively. The quantum yield ranged from 1.1 to 2.2 g E−1; the higher the incident solar radiation, the lower the quantum yield. Simultaneous photolimitation and photoinhibition of outdoor cultures was observed. The model reproduced the experimental results with less than 20% error. If photoinhibition was neglected, and a growth model that considered only photolimitation was used to fit the data, the error increased to 45%, thus reflecting the inadequacy of previous outdoor growth models that disregard photoinhibition. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 605–616, 1998.
TL;DR: The theoretical basis and quantitative evaluation of a new approach for modeling biofilm growth are presented, and the new technique looks promising for modeling diffusion-reaction-microbial growth processes in heterogeneous systems as they occur in biofilms.
Abstract: The theoretical basis and quantitative evaluation of a new approach for modeling biofilm growth are presented here. Soluble components (e.g., substrates) are represented in a continuous field, whereas discrete mapping is used for solid components (e.g., biomass). The spatial distribution of substrate is calculated by applying relaxation methods to the reaction-diffusion mass balance. A biomass density map is determined from direct integration in each grid cell of a substrate-limited growth equation. Spreading and distribution of biomass is modeled by a discrete cellular automaton algorithm. The ability of this model to represent diffusion-reaction-microbial growth systems was tested for a well-characterized system: immobilized cells growing in spherical gel beads. Good quantitative agreement with data for global oxygen consumption rate was found. The calculated concentration profiles of substrate and biomass in gel beads corresponded to those measured. Moreover, it was possible, using the discrete spreading algorithm, to predict the spatial two- and three-dimensional distribution of microorganisms in relation to, for example, substrate flux and inoculation density. The new technique looks promising for modeling diffusion-reaction-microbial growth processes in heterogeneous systems as they occur in biofilms.
TL;DR: Encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule are reported, and it is demonstrated that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks.
Abstract: A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes.
TL;DR: Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted.
Abstract: The objectives of this study were to isolate and evaluate microorganisms with the ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the presence of synthetic surfactants. Stenotrophomonas maltophilia VUN 10,010, isolated from PAH-contaminated soil, utilized pyrene as a sole carbon and energy source and also degraded other high molecular weight PAHs containing up to seven benzene rings. Various synthetic surfactants were tested for their ability to improve the PAH degradation rate of strain VUN 10,010. Anionic and cationic surfactants were highly toxic to this strain, and the Tween series was used as a growth substrate. Five nonionic surfactants (Brij 35, Igepal CA-630, Triton X-100, Tergitol NP-10, and Tyloxapol) were not utilized by, and were less toxic to, strain VUN 10,010. MSR and log Km values were determined for fluoranthene, pyrene, and benzo[a]pyrene in the presence of these nonionic surfactants and their apparent solubility was increased by a minimum of 250-fold in the presence of 10 g L−1 of all surfactants. The rate of pyrene degradation by strain VUN 10,010 was enhanced by the addition of four of the nonionic surfactants (5–10 g L−1); however, 5 g L−1 Igepal CA-630 inhibited pyrene degradation and microbial growth. The specific growth rate of VUN 10,010 on pyrene was increased by 67% in the presence of 10 g L−1 Brij 35 or Tergitol NP-10. The addition of Brij 35 and Tergitol NP-10 to media containing a single high molecular weight PAH (four and five benzene rings) as the sole carbon source increased the maximum specific PAH degradation rate and decreased the lag period normally seen for PAH degradation. The addition of Tergitol NP-10 to VUN 10,010 cultures which contained a PAH mixture (three to seven benzene rings) substantially improved the overall degradation rate of each PAH and increased the specific growth rate of VUN 10,010 by 30%. Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:482–494, 1998.
TL;DR: In this article, some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering are reviewed, and their advantages and disadvantages in different contexts are examined.
Abstract: In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides. From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities. Importantly, the range of promoter activities was covered in small steps of activity change. Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question. If the relevant variable (e.g., the flux or yield) is then measured with each of these constructs, then one can calculate the control coefficient and determine the optimal expression level. One advantage of the method is that the construct which is found to have the optimal expression level is then, in principle, ready for use in the industrial fermentation process; another advantage is that the system can be used to optimize the expression of different enzymes within the same cell.
TL;DR: The incomplete intracellular sialylation of interferon-gamma (IFN-Gamma), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intrace cellular precursor for sialic acid synthesis.
Abstract: Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the incomplete intracellular sialylation of inter- feron-g (IFN-g), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis. By intro- ducing 20 mM ManNAc into the culture medium, incom- pletely sialylated biantennary glycan structures were re- duced from 35% to 20% at the Asn 97 glycosylation site. This effect was achieved without affecting cell growth or product yield. The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltransferase, was also extracted and quantified by HPLC. Feeding of 20 mM ManNAc increased this intracellular pool of CMP-sialic acid by nearly thirtyfold compared with unsupplemented medium. When radiolabeled ManNAc was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-g as sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of product sialylation originated from the supplemental precursor. © 1998 John Wiley & Sons, Inc. Bio- technol Bioeng 58: 642-648, 1998.
TL;DR: Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening.
Abstract: Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated.
TL;DR: Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.
Abstract: This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.
TL;DR: The CO2-gradients in the culture were shown to be responsible for the limitation during the exponential growth phase, and both CO2 and light irradiance were limiting in the linear growth phase.
Abstract: The microalga Phaeodactylum tricornutum was grown in a concentric tube airlift photobioreactor. A maximum specific growth rate of 0. 023 h-1 was obtained using a superficial gas velocity around 0.055 m/s. Lower or higher gas flow rates limited the culture performance. To establish if the observed limitation was due to CO2 or to the photosynthetically active irradiance, characteristic times for mixing, mass transfer and CO2 consumption, and the photon flux absorbed by the culture were analyzed. The CO2-gradients in the culture were shown to be responsible for the limitation during the exponential growth phase, and both CO2 and light irradiance were limiting in the linear growth phase. The decrease in specific growth rate relative to the maximum was found to be related to the specific gas-liquid interfacial area, the length scale of the microeddies and the shear rate. Copyright 1998 John Wiley & Sons, Inc.
TL;DR: The competition between acetate utilizing methane-producing bacteria and sulfate-reducing bacteria (SRB) was studied in mesophilic upflow anaerobic sludge bed (UASB) reactors and the long term nature of the competition between these acetotrophs was confirmed.
Abstract: The competition between acetate utilizing methane-producing bacteria (MB) and sulfate-reducing bacteria (SRB) was studied in mesophilic (30 degrees C) upflow anaerobic sludge bed (UASB) reactors (upward velocity 1 m h-1; pH 8) treating volatile fatty acids and sulfate. The UASB reactors treated a VFA mixture (with an acetate:propionate:butyrate ratio of 5:3:2 on COD basis) or acetate as the sole substrate at different COD:sulfate ratios. The outcome of the competition was evaluated in terms of conversion rates and specific methanogenic and sulfidogenic activities. The COD:sulfate ratio was a key factor in the partitioning of acetate utilization between MB and SRB. In excess of sulfate (COD:sulfate ratio lower than 0.67), SRB became predominant over MB after prolonged reactor operation: 250 and 400 days were required to increase the amount of acetate used by SRB from 50 to 90% in the reactor treating, respectively, the VFA mixture or acetate as the sole substrate. The competition for acetate was further studied by dynamic simulations using a mathematical model based on the Monod kinetic parameters of acetate utilizing SRB and MB. The simulations confirmed the long term nature of the competition between these acetotrophs. A high reactor pH (+/-8), a short solid retention time (<150 days), and the presence of a substantial SRB population in the inoculum may considerably reduce the time required for acetate-utilising SRB to outcompete MB.
TL;DR: The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated and mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones.
Abstract: The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.
TL;DR: Mixed population biofilms consisting of Pseudomonas aeruginosa, P. fluorescens, and Klebsiella pneumoniae were grown in a flow cell under turbulent conditions with a water flow velocity of 18 cm/s and the motion of the streamers was possibly limited by the flexibility of the biofilm material.
Abstract: Mixed population biofilms consisting of Pseudomonas aeruginosa, P. fluorescens, and Klebsiella pneumoniae were grown in a flow cell under turbulent conditions with a water flow velocity of 18 cm/s (Reynolds number, Re, =1192). After 7 days the biofilms were patchy and consisted of cell clusters and streamers (filamentous structures attached to the downstream edge of the clusters) separated by interstitial channels. The cell clusters ranged in size from 25 to 750 microm in diameter. The largest clusters were approximately 85 microm thick. The streamers, which were up to 3 mm long, oscillated laterally in the flow. The motion of the streamers was recorded at various flow velocities up to 50.5 cm/s (Re 3351) using confocal scanning laser microscopy. The resulting time traces were evaluated by image analysis and fast Fourier transform analysis (FFT). The amplitude of the motion increased with flow velocity in a sigmoidal shaped curve, reaching a plateau at an average fluid flow velocity of approximately 25 cm/s (Re 1656). The motion of the streamers was possibly limited by the flexibility of the biofilm material. FFT indicated that the frequency of oscillation was directly proportional to the average flow velocity (u(ave)) below 9.5 cm/s (Re 629). At u(ave) greater than 9.5 cm/s, oscillation frequencies were above our measurable frequency range (0.12-6.7 Hz). The oscillation frequency was related to the flow velocity by the Strouhal relationship, suggesting that the oscillations were possibly caused by vortex shedding from the upstream biofilm clusters. A loss coefficient (k) was used to assess the influence of biofilm accumulation on pressure drop. The k across the flow cell colonized with biofilm was 2.2 times greater than the k across a clean flow cell.
TL;DR: Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated, and the average values of the in vitro activities during an oscillation cycle agreed well with their corresponding values for nonoscillating cultures under similar environmental conditions.
Abstract: The initial steps of glycerol dissimilation and 1,3-propanediol (1, 3-PD) formation by Klebsiella pneumoniae anaerobically grown on glycerol were studied by quantifying the in vitro and in vivo activities of enzymes in continuous culture under conditions of steady state and oscillation and during transient phases. The enzymes studied included glycerol dehydrogenase (GDH), glycerol dehydratase (GDHt), and 1,3-propanediol oxidoreductase (PDOR). Three conclusions can be drawn from the steady-state results. First, glycerol concentration in the culture is a key parameter that inversely affects the in vitro activities (concentrations) of all three enzymes, but has a positive effect on their in vivo activities. Growth rate significantly affects the ratio of in vitro and in vivo enzyme activities under low glycerol concentrations, but not under glycerol excess. Second, whereas the flux through the oxidative pathway of glycerol dissimilation is governed mainly by the regulation of in vivo enzyme activity on a metabolic level, the flux through the reductive pathway is largely controlled by the synthesis of enzymes. Third, GDHt is a major rate-liming enzyme for the consumption of glycerol and the formation of 1,3-PD in K. pneumoniae at high glycerol concentrations. Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated. The average values of the in vitro activities during an oscillation cycle agreed well with their corresponding values for nonoscillating cultures under similar environmental conditions. Experiments with step changes in the feed concentration of glycerol demonstrated that growth and product formation are very sensitive to changes of substrate concentration in the culture. This sensitivity is due to the dynamic responses of the genetic and metabolic networks. They should be considered when modeling the dynamics of the culture and attempting to improve the formation of 1,3-PD.
TL;DR: The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA), and the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with highqAb.
Abstract: Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 microM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 microM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (qAb), although they were derived from a single clone. The mu and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 microgram/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb.
TL;DR: It is found that heat input control that runs the process close to the melting and scorch temperature constraints yields faster drying times, and more uniform distributions of temperature and concentration of bound water at the end of the secondary drying stage.
Abstract: A rigorous unsteady state and spatially multidimensional model is presented and solved to describe the dynamic behavior of the primary and secondary drying stages of the lyophilization of a pharmaceutical product in vials for different operational policies. The results in this work strongly motivate the aggressive control of freeze drying and it is found that heat input control that runs the process close to the melting and scorch temperature constraints yields (i) faster drying times, and (ii) more uniform distributions of temperature and concentration of bound water at the end of the secondary drying stage.