R. Eric Berson

The Effect of Particle Size on Hydrolysis Reaction Rates and Rheological Properties in Cellulosic Slurries

The effect of varying initial particle sizes on enzymatic hydrolysis rates and rheological properties of sawdust slurries is investigated. Slurries with four particle size ranges (33 µm<x≤75 µm, 150 µm<x≤180 µm, 295 µm<x≤425 µm, and 590 µm<x≤850 µm) were subjected to enzymatic hydrolysis using an enzyme dosage of 15 filter paper units per gram of cellulose at 50°C and 250 rpm in shaker flasks. At lower initial particle sizes, higher enzymatic reaction rates and conversions of cellulose to glucose were observed. After 72 h 50 and 55% more glucose was produced from the smallest size particles than the largest size ones, for initial solids concentration of 10 and 13% (w/w), respectively. The effect of initial particle size on viscosity over a range of shear was also investigated. For equivalent initial solids concentration, smaller particle sizes result in lower viscosities such that at a concentration of 10% (w/w), the viscosity decreased from 3000 cP for 150 µm<x≤180 µm particle size slurries to 61.4 cP for 33 µm<x≤75 µm particle size slurries. Results indicate particle size reduction may provide a means for reducing the long residence time required for the enzymatic hydrolysis step in the conversion of biomass to ethanol. Furthermore, the corresponding reduction in viscosity may allow for higher solids loading and reduced reactor sizes during large-scale processing.

Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture.

Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20–60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (≤2.5 dyn/cm2) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies. Biotechnol. Bioeng. 2010; 105: 611–626.

Characterization of changes in viscosity and insoluble solids content during enzymatic saccharification of pretreated corn stover slurries

Viscosity trends in pretreated corn stover slurries undergoing enzymatic saccharification were characterized for a range of initial insoluble solids concentrations from 10% to 25% and correlated with total glucose released and changes in insoluble solids concentration throughout a 7day period. Viscosity trends are defined in two phases, which coincide with two rate zones observed in the release of sugar during enzymatic hydrolysis. Viscosity rapidly decreased as initial solids concentration decreased in the first phase, and appears to reach a steady value for the lower solids concentrations in the second phase. The first phase is defined as approximately the first 8h of the reaction based on the rates of glucose release, viscosity changes, and insoluble solids changes. A method for premixing the slurry samples in the viscometer cup prior to viscosity measurements is introduced. The method takes into consideration the need to maintain a uniform solids suspension while ensuring steady-state flow inside the viscometer cup. The slurries exhibit pseudoplastic behavior and are well described by the power law model for non-Newtonian fluids throughout the course of the reaction. Small changes in percent solids concentration lead to order of magnitude differences in viscosity.

Effects of Biaxial Oscillatory Shear Stress on Endothelial Cell Proliferation and Morphology

Wall shear stress (WSS) on anchored cells affects their responses, including cell proliferation and morphology. In this study, the effects of the directionality of pulsatile WSS on endothelial cell proliferation and morphology were investigated for cells grown in a Petri dish orbiting on a shaker platform. Time and location dependent WSS was determined by computational fluid dynamics (CFD). At low orbital speed (50 rpm), WSS was shown to be uniform (0–1 dyne/cm2) across the bottom of the dish, while at higher orbital speed (100 and 150 rpm), WSS remained fairly uniform near the center and fluctuated significantly (0–9 dyne/cm2) near the side walls of the dish. Since WSS on the bottom of the dish is two‐dimensional, a new directional oscillatory shear index (DOSI) was developed to quantify the directionality of oscillating shear. DOSI approached zero for biaxial oscillatory shear of equal magnitudes near the center and approached one for uniaxial pulsatile shear near the wall, where large tangential WSS dominated a much smaller radial component. Near the center (low DOSI), more, smaller and less elongated cells grew, whereas larger cells with greater elongation were observed in the more uniaxial oscillatory shear (high DOSI) near the periphery of the dish. Further, cells aligned with the direction of the largest component of shear but were randomly oriented in low magnitude biaxial shear. Statistical analyses of the individual and interacting effects of multiple factors (DOSI, shear magnitudes and orbital speeds) showed that DOSI significantly affected all the responses, indicating that directionality is an important determinant of cellular responses. Biotechnol. Bioeng. 2012; 109:695–707.

Computationally Determined Shear on Cells Grown in Orbiting Culture Dishes

A new computational model, using computational fluid dynamics (CFD), is presented that describes fluid behavior in cylindrical cell culture dishes resulting from motion imparted by an orbital shaker apparatus. This model allows for the determination of wall shear stresses over the entire area of the bottom surface of a dish (representing the growth surface for cells in culture) which was previously too complex for accurate quantitative analysis. Two preliminary cases are presented that show the complete spatial resolution of the shear on the bottom of the dishes. The maximum shear stress determined from the model is compared to an existing simplified point function that provides only the maximum value. Furthermore, this new model incorporates seven parameters versus the four in the previous technique, providing improved accuracy. Optimization of computational parameters is also discussed.

Detoxification of actual pretreated corn stover hydrolysate using activated carbon powder

A technique for the removal of acetic acid from an actual pretreated corn stover hydrolysate was investigated. A powdered form of activated carbon previously shown to be effective in the removal of acetic acid from a synthetic hydrolysate was utilized. The method proved to be effective at lowering acetic acid levels while exhibiting minimal adsorption of the desired sugars from the hydrolysate, although at a lower efficiency in the actual hydrolysate than in the synthetic hydrolysate. Results are obtained for temperatures between 25 and 35°C and agitation rates between 150 and 350 rpm in shake flasks. Adsorption isotherm and kinetic rate date are presented. Temperature differences over this range did not have an effect on adsorption characteristics. Five stages of detoxification were necessary to lower acetic acid concentration to the maximum 2 g/L desired for fermentation.

Deactivation of individual cellulase components

Deactivation extents of cellobiohydrolase, endoglucanase, and a total cellulase mixture (Spezyme CP) were studied independently as functions of incubating time and mixing intensity. It was found that the decrease in total cellulase activity was more strongly related to deactivation of cellobiohydrolase 1 (CBH1) than endoglucanase. The mass-averaged shear in orbiting flasks at 50, 150, and 250rpm was quantified by computational fluid dynamics and was two-orders smaller than shear in typical stirred tanks. Endoglucanase activity did not change significantly with mixing speed, but CBH1 and total cellulase activities were 10-25% higher at 250rpm compared to the lower speeds after a 24-h incubation. Total deactivation due to mechanical mixing (∼20%) may be too low to account for all the rate reduction during cellulose hydrolysis. Thermal deactivation was independent of enzyme concentration while deactivation due to mechanical stress decreased when cellulase loading increased over 0.15 filterpaperunit/ml.

Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulase.

Enzymatic hydrolysis involves complex interaction between enzyme, substrate, and the reaction environment, and the complete mechanism is still unknown. Further, glucose release slows significantly as the reaction proceeds. A model based on Langmuir binding kinetics that incorporates inactivation of adsorbed cellulase was developed that predicts product formation within 10% of experimental results for two substrates. A key premise of the model, with experimental validation, suggests that V(max) decreases as a function of time due to loss of total available enzyme as adsorbed cellulases become inactivated. Rate constants for product formation and enzyme inactivation were comparable to values reported elsewhere. A value of k(2)/K(m) that is several orders of magnitude lower than the rate constant for the diffusion-controlled encounter of enzyme and substrate, along with similar parameter values between substrates, implies a common but undefined rate-limiting step associated with loss of enzyme activity likely exists in the pathway of cellulose hydrolysis.

Spatial and temporal resolution of shear in an orbiting petri dish

It is well documented that physiological and morphological properties of anchored cells are influenced by fluid shear stress. Common orbital shakers provide a means of simultaneously applying shear stress to cells for tens to hundreds of cases by loading the shaker with multiple dishes. However, the complex flow in orbiting dishes is amenable to analytical solution for resolving shear created by the fluid motion only for simplified conditions. The only existing quantification of shear in this flow is an equation that estimates a constant scalar value of shear for the entire surface of the dish. In practice, wall shear stress (WSS) will be oscillatory rather than steady due to the travelling waveform and will vary across the surface of the dish at any instant in time. This article presents a computational model that provides complete spatial and temporal resolution of WSS over the bottom surface of a dish throughout the orbital cycle. The model is reasonably well validated by the analytical solution, with resultant WSS magnitudes that are within 0.99 ± 0.42 dyne/cm2. The model results were compared to tangential WSS magnitudes obtained using one‐dimensional optical velocimetry at discreet locations on the bottom of an orbiting dish. The experimental minimum and maximum WSS at 1 mm from the center of the dish were 6 and 7 dyne/cm2, respectively, whereas WSS generated from the computational model ranged from 0.5 to 8.5 dyne/cm2. The experimental minimum and maximum WSS at 12 mm from the center of the dish were 6 and 16 dyne/cm2, respectively, whereas WSS generated from the computational model ranged from 0.5 to 14 dyne/cm2. Discrepancies between the experimental and computational data may be attributed to a sparse sampling rate for the experimental probe, a sharp gradient at the sample area which could cause the unidirectional probe to be inaccurate if its location were not precise, and too few particles to track and a scattering of the signal by the free surface when the liquid is shallow.

Enhanced mixing and mass transfer in a recirculation loop results in high cell densities in a roller bottle reactor.

A recirculation loop added to a large‐scale roller bottle reactor resulted in high cell densities as compared to standard roller bottles. Four different mammalian cell lines reached an average maximum density equal to 5.4 × 106 cells /mL (σ = 0.263), which was between 2.13 and 2.95 times greater than the densities in roller bottles without recirculation using the same cell lines. The high densities were maintained over long durations (>25 days) while the reactor operated with continuous perfusion. The increased densities are attributed to enhanced liquid mixing and oxygen transfer that occur as a result of the recirculation loop. Models were developed that describe axial liquid flow and oxygen transfer in both the sample loop and the reactor growth chamber. Axial dispersion and oxygen transfer coefficients are presented for a variety of operating conditions. The increased oxygen transfer characteristics of the reactor allow for easy scale‐up of roller bottle cultures by operating at larger volumes with greater liquid depths than conventional roller bottles permit. The surface‐area‐to‐volume ratio in the tests performed was 0.206 versus 1.16 cm−1 in a standard roller bottle.