Showing papers in "Biotechnology and Bioengineering in 2002"
TL;DR: This work presents a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway, and offers the possibility of studying important questions regarding the stability and control of metabolic fluxes.
Abstract: Application of metabolic engineering principles to the rational design of microbial production processes crucially depends on the ability to describe quantitatively the systemic behavior of the central carbon metabolism to redirect carbon fluxes to the product-forming pathways. Despite the importance for several production processes, development of an essential dynamic model for central carbon metabolism of Escherichia coli has been severely hampered by the current lack of kinetic information on the dynamics of the metabolic reactions. Here we present the design and experimental validation of such a dynamic model, which, for the first time, links the sugar transport system (i.e., phosphotransferase system [PTS]) with the reactions of glycolysis and the pentose-phosphate pathway. Experimental observations of intracellular concentrations of metabolites and cometabolites at transient conditions are used to validate the structure of the model and to estimate the kinetic parameters. Further analysis of the detailed characteristics of the system offers the possibility of studying important questions regarding the stability and control of metabolic fluxes. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 53–73; 2002.
574 citations
TL;DR: It is observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability.
Abstract: We describe the design, fabrication, and performance of a bioreactor that enables both morphogenesis of 3D tissue structures under continuous perfusion and repeated in situ observation by light microscopy. Three-dimensional scaffolds were created by deep reactive ion etching of silicon wafers to create an array of channels (through-holes) with cell-adhesive walls. Scaffolds were combined with a cell-retaining filter and support in a reactor housing designed to deliver a continuous perfusate across the top of the array and through the 3D tissue mass in each channel. Reactor dimensions were constructed so that perfusate flow rates meet estimated values of cellular oxygen demands while providing fluid shear stress at or below a physiological range (<2 dyne cm(2)), as determined by comparison of numerical models of reactor fluid flow patterns to literature values of physiological shear stresses. We studied the behavior of primary rat hepatocytes seeded into the reactors and cultured for up to 2 weeks, and found that cells seeded into the channels rearranged extensively to form tissue like structures and remained viable throughout the culture period. We further observed that preaggregation of the cells into spheroidal structures prior to seeding improved the morphogenesis of tissue structure and maintenance of viability. We also demonstrate repeated in situ imaging of tissue structure and function using two-photon microscopy.
495 citations
TL;DR: Cell seeding within porous alginate scaffolds is addressed in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution, to enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.
Abstract: Cardiac tissue engineering has evolved as a potential therapeutic approach to assist in cardiac regeneration. We have recently shown that tissue-engineered cardiac graft, constructed from cardiomyocytes seeded within an alginate scaffold, is capable of preventing the deterioration in cardiac function after myocardial infarction in rats. The present article addresses cell seeding within porous alginate scaffolds in an attempt to achieve 3D high-density cardiac constructs with a uniform cell distribution. Due to the hydrophilic nature of the alginate scaffold, its >90% porosity and interconnected pore structure, cell seeding onto the scaffold was efficient and short, up to 30 min. Application of a moderate centrifugal force during cell seeding resulted in a uniform cell distribution throughout the alginate scaffolds, consequently enabling the loading of a large number of cells onto the 3D scaffolds. The percent cell yield in the alginate scaffolds ranged between 60-90%, depending on cell density at seeding; it was 90% at seeding densities of up to 1 x 10(8) cells/cm(3) scaffold and decreased to 60% at higher densities. The highly dense cardiac constructs maintained high metabolic activity in culture. Scanning electron microscopy revealed that the cells aggregated within the scaffold pores. Some of the aggregates were contracting spontaneously within the matrix pores. Throughout the culture there was no indication of cardiomyocyte proliferation within the scaffolds, nor was it found in 3D cultures of cardiofibroblasts. This may enable the development of cardiac cocultures, without domination of cardiofibroblasts with time.
410 citations
TL;DR: Compared four standard ES cell differentiation culture systems are compared by measuring aspects of embryoid body formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays.
Abstract: Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.
377 citations
TL;DR: The more precise rate coefficients of the first-order model provided a better means than the Monod parameters, to quantitatively compare the reduction rates between metals, electron donors, and DMRB species.
Abstract: The reduction kinetics of Fe(III)citrate, Fe(III)NTA, Co(III)EDTA-, U(VI)O(2) (2+), Cr(VI)O(4) (2-), and Tc(VII)O(4) (-) were studied in cultures of dissimilatory metal reducing bacteria (DMRB): Shewanella alga strain BrY, Shewanella putrefaciens strain CN32, Shewanella oneidensis strain MR-1, and Geobacter metallireducens strain GS-15. Reduction rates were metal specific with the following rate trend: Fe(III)citrate > or = Fe(III)NTA > Co(III)EDTA- >> UO(2)(2+) > CrO(4)(2-) > TcO(4)(-), except for CrO(4) (2-) when H(2) was used as electron donor. The metal reduction rates were also electron donor dependent with faster rates observed for H(2) than lactate- for all Shewanella species despite higher initial lactate (10 mM) than H2 (0.48 mM). The bioreduction of CrO(4) (2-) was anomalously slower compared to the other metals with H(2) as an electron donor relative to lactate and reduction ceased before all the CrO(4)(2-) had been reduced. Transmission electron microscopic (TEM) and energy-dispersive spectroscopic (EDS) analyses performed on selected solids at experiment termination found precipitates of reduced U and Tc in association with the outer cell membrane and in the periplasm of the bacteria. The kinetic rates of metal reduction were correlated with the precipitation of reduced metal phases and their causal relationship discussed. The experimental rate data were well described by a Monod kinetic expression with respect to the electron acceptor for all metals except CrO(4)(2-), for which the Monod model had to be modified to account for incomplete reduction. However, the Monod models became statistically over-parameterized, resulting in large uncertainties of their parameters. A first-order approximation to the Monod model also effectively described the experimental results, but the rate coefficients exhibited far less uncertainty. The more precise rate coefficients of the first-order model provided a better means than the Monod parameters, to quantitatively compare the reduction rates between metals, electron donors, and DMRB species.
338 citations
TL;DR: A facile methodology has been developed for covalently derivatizing the surfaces of common materials with a designed antibacterial polycation, poly(vinyl-N-pyridinium bromide), wherein the first, key step involves surface coating with a nanolayer of silica.
Abstract: A facile methodology has been developed for covalently derivatizing the surfaces of common materials with a designed antibacterial polycation, poly(vinyl-N-pyridinium bromide), wherein the first, key step involves surface coating with a nanolayer of silica. Various commercial synthetic polymers derivatized in this manner become bactericidal-they kill up to 99% of deposited, from either an aerosol or an aqueous suspension, Gram-positive and Gram-negative bacteria on contact.
330 citations
TL;DR: Cadmium sulfide nanoparticles were synthesized intracellularly by a Schizosaccharomyces pombe strain when challenged with 1 mM cadmium in solution and exhibited an absorbance maximum at 305 nm.
Abstract: Cadmium sulfide nanoparticles were synthesized intracellularly by a Schizosaccharomyces pombe strain when challenged with 1 mM cadmium in solution. The nanoparticles, a known semiconducting material, exhibited an absorbance maximum at 305 nm. X-ray scattering data showed that the nanoparticles had a Wurtzite (Cd16S20)-type hexagonal lattice structure and most of the nanoparicles were in the size range of 1–1.5 nm. The nanoparticles were used in the fabrication of a heterojunction with poly (p-phenylenevinylene). The diode exhibited ∼75 mA/cm2 current at 10 V when forward biased and the breakdown occurred at ∼15 V in the reverse biased mode. These characteristics are considered ideal for a diode. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 583–588, 2002.
329 citations
TL;DR: A mathematical model describing the constitutive properties of biofilms is required for predicting biofilm deformation, failure, and detachment in response to mechanical forces and a system of vis coelastic fluid equations with a linear Jeffreys viscoelastic stress-strain law is presented here.
Abstract: A mathematical model describing the constitutive properties of biofilms is required for predicting biofilm deformation, failure, and detachment in response to mechanical forces. Laboratory observations indicate that biofilms are viscoelastic materials. Likewise, current knowledge of biofilm internal structure suggests modeling biofilms as associated polymer viscoelastic systems. Supporting experimental results and a system of viscoelastic fluid equations with a linear Jeffreys viscoelastic stress-strain law are presented here. This system of equations is based on elements of associated polymer physics and is also consistent with presented and previous experimental results. A number of predictions can be made. One particularly interesting result is the prediction of an elastic relaxation time on the order of a few minutes-biofilm disturbances on shorter time scales produce an elastic response, biofilm disturbances on longer time scales result in viscous flow, i.e., nonreversible biofilm deformation. Although not previously recognized, evidence of this phenomenon is in fact present in recent experimental results.
317 citations
TL;DR: It is demonstrated that hydrogen-producing acidogenic sludge could agglutinate into granules in a well-mixed reactor treating a synthetic sucrose-containing wastewater at 26 degrees C, pH 5.5, with 6 h of hydraulic retention.
Abstract: This study demonstrated that hydrogen-producing acidogenic sludge could agglutinate into granules in a well-mixed reactor treating a synthetic sucrose-containing wastewater at 26°C, pH 5.5, with 6 h of hydraulic retention. A typical matured granule is 1.6 mm in diameter, 1.038 g/mL in density, 11% in ash content, and over 50 m/h in settling velocity. Treating a solution containing 12.15 g/L of sucrose at a volumetric loading rate of 48.6 g/(L · d), the reactor containing 20 g/L of granular sludge degraded 97% of sucrose. Effluent comprised 46% acetate and 49% butyrate and the methane-free biogas comprised 63% hydrogen, 35% carbon dioxide, and 2% nitrogen. Hydrogen production rate was 13.0 L/(L · d), and the yield was 0.28 L/g-sucrose. The granule had multiple cracks on the surface and comprised two morphological types of bacteria: fusiform bacilli and a spore-forming bacterium. Phylogenetic analysis showed that 69.1% of the clones were affiliated with four Clostridium species in the family Clostridiaceae, and 13.5% with Sporolactobacillus racemicus in the Bacillus/Staphylococcus group. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 44–52, 2002; DOI 10.1002/bit.10174
291 citations
TL;DR: It is demonstrated that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H2-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions.
Abstract: The green alga, Chlamydomonas reinhardtii, is capable of sustained H2 photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O2-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10–15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H2 gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O2-consumption phase, an anaerobic phase, a H2-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H2-production phase; (3) re-addition of small quantities of sulfate (12.5–50 μM MgSO4, final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H2 production, an increase in the length of the H2-production phase, and an increase in the total amount of H2 produced; and (4) increases in the culture optical density in the presence of 50 μM sulfate result in a decrease in the initial specific rates of H2 production and in an earlier start of the H2-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H2 production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H2-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 731–740, 2002.
279 citations
TL;DR: Results show that acid catalyzed hydrolysis is proportional to H+ concentration, and overall yields of glucose obtained from cellobiose and cellulose are higher for the best carboxylic acid tested, maleic acid, when compared to sulfuric acid at equivalent solution pH.
Abstract: Cellulolytic enzymes consist of a catalytic domain, a linking peptide, and a binding domain. The paper describes research on carboxylic acids that have potential as catalytic domains for constructing organic macromolecules for use in cellulose hydrolysis that mimic the action of enzymes. The tested domains consist of the series of mono-, di-, and tricarboxylic acids with a range of pK(a)'s. This paper systematically characterizes the acids with respect to hydrolysis of cellobiose, cellulose in biomass, and degradation of glucose and compares these kinetics data to dilute sulfuric acid. Results show that acid catalyzed hydrolysis is proportional to H+ concentration. The tested carboxylic acids did not catalyze the degradation of glucose while sulfuric acid catalyzed the degradation of glucose above that of water alone. Consequently, overall yields of glucose obtained from cellobiose and cellulose are higher for the best carboxylic acid tested, maleic acid, when compared to sulfuric acid at equivalent solution pH.
TL;DR: Pd, and also Pt and Rh, could be recovered by resting cell suspensions under H2 from an industrial processing wastewater, suggesting a possible future application of bioprocessing technology for precious metals.
Abstract: The reduction of Pd(II) to Pd(0) was accelerated by using the sulfate-reducing bacterium Desulfovibrio desulfuricans NCIMB 8307 at the expense of formate or H(2) as electron donors at pH 2-7. With formate no reduction occurred at pH 2, but with H(2) 50% of the activity was retained at pH 2, with the maximum rate (1.3-1.4 micromol min(-1) mg dry cells(-1)) seen at pH 3-7, which was similar to the rate with formate at neutral pH. Excess nitrate was inhibitory to Pd(II) reduction using formate, but not H(2). Chloride ion was inhibitory as low as 100 mM using formate but with H(2) only ca. 25% inhibition was observed at 500 mM Cl(-) and H(2) was concluded to be the electron donor of choice for the potential remediation of industrial wastes. Deposited Pd was visible on the cells using transmission and scanning electron microscopy and analysis by energy dispersive X-ray microanalysis (EDAX) identified the deposit as Pd, confirmed as Pd(0) by X-ray powder diffraction analysis (XRD). The crystal size of the biodeposited Pd(0) was determined to be only 50% of the size of Pd(0) crystals manufactured chemically from Pd(II) at the expense of H(2) and, unlike the chemically manufactured material, the biocrystal size was independent of the pH. The "biological" Pd(0) functioned as a superior chemical catalyst in a test reaction which liberated hydrogen from hypophosphite. Pd, and also Pt and Rh, could be recovered by resting cell suspensions under H(2) from an industrial processing wastewater, suggesting a possible future application of bioprocessing technology for precious metals.
TL;DR: The total concentrations of electrostatic binding sites found in this study were 20- to 30-fold of those reported foracterial cell surface, implying that EPS might be more crucial in biosorption of metals than bacterial cell surface in wastewater treatment and in bioremediation.
Abstract: Electrostatic binding sites of extracellular polymeric substances (EPS) were characterized from titration data using linear programming analysis. Test results for three synthetic solutions of given solutes comprising amino, carboxyl, and phenolic groups indicated that this method was able to identify the electrostatic binding sites. For the six sites with pK(a) between 3 and 10, the estimated pK(a) deviated 0.11 +/- 0.09 from the theoretical values, and the estimated concentrations deviated 3.0% +/- 0.9% from the actual concentrations. Two EPS samples were then extracted from a hydrogen-producing sludge (HPS) and a sulfate-reducing biofilm (SRB). Analysis of charge excess data in titration from pH 3 to 11 indicated that the EPS of HPS comprised of five electrostatic binding sites with pK(a) ranging from 3 to 11. The pK(a) values of these binding sites and the possible corresponding functional groups were pK(a) 4.8 (carboxyl), pK(a) 6.0 (carboxyl/phosphoric), pK(a) 7.0 (phosphoric), pK(a) 9.8 (amine/phenolic), and pK(a) 11.0 (hydroxyl). EPS of the SRB comprised five of similar binding sites (with corresponding pK(a) values of 4.4, 6.0, 7.4, 9.4, and 11.0), plus one extra site at pK(a) 8.2, which was likely corresponding to the sulfhydryl group. The total electrostatic binding site concentration of EPS extracted from HPS were 10.88 mmol/g-EPS, of which the highest concentration was from the site of pK(a) 11.0. The corresponding values for the EPS extracted from SRB were 16.44 mmol/g-EPS and pK(a) 4.4. The total concentrations of electrostatic binding sites found in this study were 20- to 30-fold of those reported for bacterial cell surface, implying that EPS might be more crucial in biosorption of metals than bacterial cell surface in wastewater treatment and in bioremediation.
TL;DR: It can be concluded that a one-stage ammonium removal process is probably not optimal if a good nitrogen effluent is required, and a two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.
Abstract: A mathematical model for nitrification and anaerobic ammonium oxidation (ANAMMOX) processes in a single biofilm reactor (CANON) was developed. This model describes completely autotrophic conversion of ammonium to dinitrogen gas. Aerobic ammonium and nitrite oxidation were modeled together with ANAMMOX. The sensitivity of kinetic constants and biofilm and process parameters to the process performance was evaluated, and the total effluent concentrations were, in general, found to be insensitive to affinity constants. Increasing the amount of biomass by either increasing biofilm thickness and density or decreasing porosity had no significant influence on the total effluent concentrations, provided that a minimum total biomass was present in the reactor. The ANAMMOX process always occurred in the depth of the biofilm provided that the oxygen concentration was limiting. The optimal dissolved oxygen concentration level at which the maximum nitrogen removal occurred is related to a certain ammonium surface load on the biofilm. An ammonium surface load of 2 g N/m2. d, associated with a dissolved oxygen concentration level of 1.3 g O2/m3 in the bulk liquid and with a minimum biofilm depth of 1 mm seems a proper design condition for the one-stage ammonium removal process. Under this condition, the ammonium removal efficiency is 94% (82% for the total nitrogen removal efficiency) (30 degrees C). Better ammonium removal could be achieved with an increase in the dissolved oxygen concentration level, but this would strongly limit the ANAMMOX process and decrease total nitrogen removal. It can be concluded that a one-stage process is probably not optimal if a good nitrogen effluent is required. A two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.
TL;DR: It is found that pO(2) values below 11 Torr might impair proper tissue development in the center, and the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures.
Abstract: Nutrient and oxygen supply of cells are crucial to tissue engineering in general. If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely. Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO2) in culture media or described the use of invasive techniques with spatially limited resolution. The experimental setup described here allows for continuous, noninvasive, high-resolution pO2 measurements over the cross-section of cultivated tissues. Applying a recently developed technique for time-resolved pO2 sensing using optical sensor foils, containing luminescent O2-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period. Cylindrical tissue samples were immobilized on top of the sensors. By measuring the luminescence decay time, two-dimensional pO2 distributions across the tissue section in contact with the foil surface were determined. We applied this technique to cartilage explants and to tissue-engineered cartilage. For both tissue types, changes were detected in monotonously decreasing gradients of pO2 from the surface with high pO2 to minimum pO2 values in the center of the samples. Nearly anoxic conditions were observed in tissue constructs (∼0 Torr) but not in excised cartilage discs (∼20 Torr) after 1 day. Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function. Additionally, histological analysis revealed a maximum depth of ∼1.3 mm of regular cartilage development in constructs grown under the applied culture conditions. Correlating analytical and histological analysis with the oxygen distributions, we found that pO2 values below 11 Torr might impair proper tissue development in the center. The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 73–83, 2002.
TL;DR: It is shown that all isotopes of each individual element must, however, be corrected in one single step and that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.
Abstract: In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of (13)C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.
TL;DR: Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system.
Abstract: Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.
TL;DR: The bacterial consortium MPD‐M, isolated from sediment associated with Colombian mangrove roots, was effective in the treatment of hydrocarbons in water with salinities varying from 0 to 180 g L−1 and biodegradation was evident even at the highest salinity evaluated.
Abstract: The bacterial consortium MPD-M, isolated from sediment associated with Colombian mangrove roots, was effective in the treatment of hydrocarbons in water with salinities varying from 0 to 180 g L -1 . Where the salinity of the culture medium surpassed 20 g L -1 , its effectiveness increased when the cells were immobilized on polypropylene fibers. Over the range of salinity evaluated, the immobilized cells significantly enhanced the biodegradation rate of crude oil compared with free-living cells, especially with increasing salinity in the culture medium. Contrary to that observed in free cell systems, the bacterial consortium MPD-M was highly stable in immobilized systems and it was not greatly affected by increments in salinity. Biodegradation was evident even at the highest salinity evaluated (180 g L -1 ), where biodegradation was between 4 and 7 times higher with immobilized cells compared to free cells. The biodegradation of pristane (PR) and phytane (PH) and of the aromatic fraction was also increased using cells immobilized on polypropylene fibers.
TL;DR: Protein-protein interactions were measured for ovalbumin and for lysozyme in aqueous salt solutions to correlated with a proposed potential of mean force equal to the free energy to desolvate the protein surface that is made inaccessible to the solvent due to the protein-protein interaction.
Abstract: Protein-protein interactions were measured for ovalbumin and for lysozyme in aqueous salt solutions. Protein-protein interactions are correlated with a proposed potential of mean force equal to the free energy to desolvate the protein surface that is made inaccessible to the solvent due to the protein-protein interaction. This energy is calculated from the surface free energy of the protein that is determined from protein-salt preferential-interaction parameter measurements. In classical salting-out behavior, the protein-salt preferential interaction is unfavorable. Because addition of salt raises the surface free energy of the protein according to the surface-tension increment of the salt, protein-protein attraction increases, leading to a reduction in solubility. When the surface chemistry of proteins is altered by binding of a specific ion, salting-in is observed when the interactions between (kosmotrope) ion-protein complexes are more repulsive than those between the uncomplexed proteins. However, salting-out is observed when interactions between (chaotrope) ion-protein complexes are more attractive than those of the uncomplexed proteins.
TL;DR: The production of galacto‐oligosaccharides from lactose by A. oryzae β‐galactosidase immobilized on cotton cloth was studied and found that more and larger GOS can be produced with higher initial lactose concentrations.
Abstract: The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.
TL;DR: Furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose, and this activity was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.
Abstract: The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.
TL;DR: The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells.
Abstract: A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.
TL;DR: These conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.
Abstract: A comparison of the effects of laminar versus turbulent flow regime on the characteristics of a single-species biofilm is presented. The study was carried out by growing Pseudomonas fluorescens biofilms in a flow cell and studying the different layers of the biological matrix with a confocal laser-scanning microscope. The following conclusions were obtained: i) a higher concentration of cells was found in the upper layers of the microbial films than in their inner layers, regardless of the flow regime; ii) the fraction of cells in the overall biofilm mass decreased with time as the film grew; and iii) under laminar flow the total number of cells was higher than in biofilms formed under turbulent flow, but the latter had a higher number of cells per unit volume. Such conclusions, together with the fact that the biofilms were more dense and stable when formed in contact with turbulent flows, favor the design of more compact and efficient biofilm reactors operating in turbulent conditions.
TL;DR: The use of FBA is broadened to map the optimal metabolic flux distribution onto a single plane, which is defined by the availability of two key substrates, and a “phase plane” analysis of the metabolic genotype–phenotype relation relevant for a range of growth conditions is given.
Abstract: Genome-scale metabolic maps can be reconstructed from annotated genome sequence data, biochemical literature, bioinformatic analysis, and strain-specific information. Flux-balance analysis has been useful for qualitative and quantitative analysis of metabolic reconstructions. In the past, FBA has typically been performed in one growth condition at a time, thus giving a limited view of the metabolic capabilities of a metabolic network. We have broadened the use of FBA to map the optimal metabolic flux distribution onto a single plane, which is defined by the availability of two key substrates. A finite number of qualitatively distinct patterns of metabolic pathway utilization were identified in this plane, dividing it into discrete phases. The characteristics of these distinct phases are interpreted using ratios of shadow prices in the form of isoclines. The isoclines can be used to classify the state of the metabolic network. This methodology gives rise to a "phase plane" analysis of the metabolic genotype-phenotype relation relevant for a range of growth conditions. Phenotype phase planes (PhPPs) were generated for Escherichia coli growth on two carbon sources (acetate and glucose) at all levels of oxygenation, and the resulting optimal metabolic phenotypes were studied. Supplementary information can be downloaded from our website (http://epicurus.che.udel.edu).
TL;DR: A technique to measure the pH environment of delivered DNA, from which the degree to which vectors avoided trafficking to acidic Iysosomes could be determined is developed.
Abstract: The degradation of DNA in lysosomes represents a major obstacle to efficient nonviral gene delivery. The rational design of vectors that overcome this obstacle requires a better understanding of the lysosomal barrier to gene delivery, which in turn requires a means to investigate this intermediate step. To this end, we developed a technique to measure the pH environment of delivered DNA, from which the degree to which vectors avoided trafficking to acidic Iysosomes could be determined. The measured average pH of DNA delivered using poly-L-lysine (PLL) polyplexes was 4.5, suggesting that PLL polyplexes were trafficked to acidic lysosomes. Other vectors could avoid or buffer the pH of Iysosomes as DNA delivered using Lipofectamine Plus, polyethylenimine (PEI), linear polyethylenimine (LPEI), and two degradable poly(beta-amino ester)s (poly-1 and poly-2) had average pH values of 7.1, 5.9, 5.0, 6.7, and 6.4, respectively.
TL;DR: The enzymatic digestibility of steam-exploded Douglas-fir wood chips was significantly improved using an optimized alkaline peroxide treatment, leaving a cellulose-rich residue that was completely hydrolyzed within 48 h, using an enzyme loading of 10 FPU/g cellulose.
Abstract: The enzymatic digestibility of steam-exploded Douglas-fir wood chips (steam exploded at 195°C, 4.5 min, and 4.5% (w/w) SO2) was significantly improved using an optimized alkaline peroxide treatment. Best hydrolysis yields were attained when the steam-exploded material was post-treated with 1% hydrogen peroxide at pH 11.5 and 80°C for 45 min. This alkaline peroxide treatment was applied directly to the water-washed, steam-exploded material eliminating the need for independent alkali treatment with 0.4% NaOH, which has been traditionally used to post-treat wood samples to try to remove residual lignin. Approximately 90% of the lignin in the original wood was solubilized by this novel procedure, leaving a cellulose-rich residue that was completely hydrolyzed within 48 h, using an enzyme loading of 10 FPU/g cellulose. About 82% of the originally available polysaccharide components of the wood could be recovered. The 18% of the carbohydrate that was not recovered was lost primarily to sugar degradation during steam explosion. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 678–684, 2002; DOI 10.1002/bit.10159
TL;DR: The results indicate that all four cell lines are able to withstand relatively intense energy dissipation rates, which is orders of magnitude higher than the maximum local energy Dissipation rates generated by impellers in bioreactors, but comparable to that associated with small bursting bubbles.
Abstract: A microfluidic device was fabricated via photolithographic techniques which can create transient elongational and shear forces ranging over three orders of magnitude while still maintaining laminar flow conditions. The contractional fluid flow inside the microfluidic device was simulated with FLUENT (a computational fluid dynamics computer program) and the local deformation forces were characterized with the scalar quantity, local energy dissipation rate. The sensitivities of four cell lines (CHO, HB-24, Sf-9, and MCF7) were tested in the device. The results indicate that all four cell lines are able to withstand relatively intense energy dissipation rates (up to 104–105 kW/m3), which is orders of magnitude higher than the maximum local energy dissipation rates generated by impellers in bioreactors, but comparable to that associated with small bursting bubbles. While the concept that suspended animal cells are relatively robust with respect to purely hydrodynamic forces in bioprocess equipment is well known, these results quantitatively demonstrate these observations. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 428–437, 2002.
TL;DR: It is demonstrated that treatment of porcine small intestine with a peracetic acid/ethanol solution leads to a virus‐free, non‐crosslinked biomaterial safe for xenotransplantation into humans.
Abstract: The goal of this study was to evaluate the efficacy of a virus-inactivating process for use during the preparation of porcine-derived extracellular matrix biomaterials for human clinical implantation. Porcine small intestine, the source material for the tissue-engineered, small intestinal submucosa (SIS) biomaterial, was evaluated. Relevant enveloped, non-enveloped, and model viruses representative of different virus families were included in the investigation: porcine parvovirus (PPV), porcine reovirus, murine leukemia retrovirus (LRV), and porcine pseudorabies (herpes) virus (PRV). Samples of small intestine were deliberately inoculated with approximately 1 x 10(7) plaque-forming units (PFU) of virus which were thereafter exposed to a 0.18% peracetic acid/4.8% aqueous ethanol mixture for time periods ranging from 5 minutes to 2 hours. Enveloped viruses were more easily inactivated than non-enveloped viruses, but material processed for 30 minutes or longer inactivated all of the viruses. D(10) values were calculated and used to extrapolate the extent of inactivation after 2 hours. Viral titers were reduced by more than 14.0 log(10) PPV, 21.0 log(10) reovirus, 40.0 log(10) PRV, and 27.0 log(10) LRV, meeting international standards for viral sterility. These results demonstrate that treatment of porcine small intestine with a peracetic acid/ethanol solution leads to a virus-free, non-crosslinked biomaterial safe for xenotransplantation into humans.
TL;DR: The control of oxygen concentration in cell microenvironment can improve the structure and function of engineered cardiac muscle and form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.
Abstract: Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., ∼100 μm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter × 2 mm thick, made of polyglycolic acid, 24 × 106 cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1–8), each containing one construct directly perfused with culture medium at a flow rate of 0.2–3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric α-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 617–625, 2002.
TL;DR: Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp.
Abstract: Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W · L−1, which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 33–41, 2002.