Mixing design for enzymatic hydrolysis of sugarcane bagasse: methodology for selection of impeller configuration

One of the major process bottlenecks for viable industrial production of second generation ethanol is related with technical–economic difficulties in the hydrolysis step. The development of a methodology to choose the best configuration of impellers towards improving mass transfer and hydrolysis yield together with a low power consumption is important to make the process cost-effective. In this work, four dual impeller configurations (DICs) were evaluated during hydrolysis of sugarcane bagasse (SCB) experiments in a stirred tank reactor (3 L). The systems tested were dual Rushton turbine impellers (DIC1), Rushton and elephant ear (down-pumping) turbines (DIC2), Rushton and elephant ear (up-pumping) turbines (DIC3), and down-pumping and up-pumping elephant ear turbines (DIC4). The experiments were conducted during 96 h, using 10 % (m/v) SCB, pH 4.8, 50 °C, 10 FPU/g_biomass, 470 rpm. The mixing time was successfully used as the characteristic parameter to select the best impeller configuration. Rheological parameters were determined using a rotational rheometer, and the power consumptions of the four DICs were on-line measured with a dynamometer. The values obtained for the energetic efficiency (the ratio between the cellulose to glucose conversion and the total energy) showed that the proposed methodology was successful in choosing a suitable configuration of impellers, wherein the DIC4 obtained approximately three times higher energetic efficiency than DIC1. Furthermore a scale-up protocol (factor scale-up 1000) for the enzymatic hydrolysis reactor was proposed.     

Mass transfer and mixing rates in fermentation vessels

The effects on mass-transfer and overall mixing rates of varying impeller geometry and operating speed have been studied for flat-bladed turbines in laboratory fermentors, in aerated aqueous solutions, and in unaerated and aerated suspensions (1.6% w/v) of paper pulp. In the absence of suspended solid, oxygen absorption rates could be correlated directly with power input. In the pulp suspension, oxygen absorption at a given power input was influenced by impeller geometry and operating speed. The data for the three-phase system can be correlated by a dimensionless equation relating oxygen-transfer rates and mixing times to the geometrical and operating parameters of the impellers.     

A comprehensive comparison of mixing, mass transfer, Chinese hamster ovary cell growth, and antibody production using Rushton turbine and marine impellers

Large scale production of monoclonal antibodies has been accomplished using bioreactors with different length to diameter ratios, and diverse impeller and sparger designs. The differences in these physical attributes often result in dissimilar mass transfer, mechanical stresses due to turbulence and mixing inside the bioreactor that may lead to disparities in cell growth and antibody production. A rational analysis of impeller design parameters on cell growth, protein expression levels and subsequent antibody production is needed to understand such differences. The purpose of this study was to examine the impact of Rushton turbine and marine impeller designs on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production and quality. Experiments to evaluate mass transfer and mixing characteristics were conducted to determine if the nutrient requirements of the culture would be met. The analysis of mixing times indicated significant differences between marine and Rushton turbine impellers at the same power input per unit volume of liquid (P/V). However, no significant differences were observed between the two impellers at constant P/V with respect to oxygen and carbon dioxide mass transfer properties. Experiments were conducted with CHO cells to determine the impact of different flow patterns arising from the use of different impellers on cell growth, metabolism and antibody production. The analysis of cell culture data did not indicate any significant differences in any of the measured or calculated variables between marine and Rushton turbine impellers. More importantly, this study was able to demonstrate that the quality of the antibody was not altered with a change in the impeller geometry.     

Torque measurements reveal large process differences between materials during high solid enzymatic hydrolysis of pretreated lignocellulose

ABSTRACT: BACKGROUND: A common trend in the research on 2nd generation bioethanol is the focus on intensifying the process and increasing the concentration of water insoluble solids (WIS) throughout the process. However, increasing the WIS content is not without problems. For example, the viscosity of pretreated lignocellulosic materials is known to increase drastically with increasing WIS content. Further, at elevated viscosities, problems arise related to poor mixing of the material, such as poor distribution of the enzymes and/or difficulties with temperature and pH control, which results in possible yield reduction. Achieving good mixing is unfortunately not without cost, since the power requirements needed to operate the impeller at high viscosities can be substantial. This highly important scale-up problem can easily be overlooked. RESULTS: In this work, we monitor the impeller torque (and hence power input) in a stirred tank reactor throughout high solid enzymatic hydrolysis (< 20% WIS) of steam-pretreated Arundo donax and spruce. Two different process modes were evaluated, where either the impeller speed or the impeller power input was kept constant. Results from hydrolysis experiments at a fixed impeller speed of 10 rpm show that a very rapid decrease in impeller torque is experienced during hydrolysis of pretreated arundo (i.e. it loses its fiber network strength), whereas the fiber strength is retained for a longer time within the spruce material. This translates into a relatively low, rather WIS independent, energy input for arundo whereas the stirring power demand for spruce is substantially larger and quite WIS dependent. By operating the impeller at a constant power input (instead of a constant impeller speed) it is shown that power input greatly affects the glucose yield of pretreated spruce whereas the hydrolysis of arundo seems unaffected. CONCLUSIONS: The results clearly highlight the large differences between the arundo and spruce materials, both in terms of needed energy input, and glucose yields. The impact of power input on glucose yield is furthermore shown to vary significantly between the materials, with spruce being very affected while arundo is not. These findings emphasize the need for substrate specific process solutions, where a short pre-hydrolysis (or viscosity reduction) might be favorable for arundo whereas fed-batch might be a better solution for spruce. RESULTS: In this work, we monitor the impeller torque (and hence power input) in a stirred tank reactor throughout high solid enzymatic hydrolysis (< 20% WIS) of steam-pretreated Arundo donax and spruce. Results from hydrolysis experiments at a stirrer speed of 10 rpm show that a very rapid decrease in impeller torque is experienced during hydrolysis of pretreated arundo (i.e. it loses its fiber network strength), whereas the fiber strength is retained for a longer time within the spruce material. This translates into a relatively low, rather WIS independent, energy input for arundo whereas the stirring power demand for spruce is substantially larger and quite WIS dependent. By operating the impeller at a constant power input (instead of a constant impeller speed) it is shown that power input greatly affects the hydrolysis yield of pretreated spruce whereas the hydrolysis of arundo seems unaffected. CONCLUSIONS: The results clearly highlight the large differences between the two materials, both in terms of needed energy input, and hydrolysis yields. The impact of power input is furthermore shown to vary significantly between the materials, with spruce being very affected while arundo is not. These findings emphasize the need for substrate specific process solutions, where a short pre-hydrolysis (or viscosity reduction) might be favorable for arundo whereas fed-batch might be a better solution for spruce.     

CFD simulation of mixing for high-solids anaerobic digestion

A computational fluid dynamics (CFD) model that simulates mechanical mixing for high-solids anaerobic digestion was developed. Numerical simulations of mixing manure slurry which exhibits non-Newtonian pseudo-plastic fluid behavior were performed for six designs: (i) one helical ribbon impeller; (ii) one anchor impeller; (iii) one curtain-type impeller; (iv) three counterflow (CF-2) impellers; (v) two modified high solidity (MHS 3/39°) impellers; and (vi) two pitched blade turbine impellers. The CFD model was validated against measurements for mixing a Herschel-Bulkley fluid by ribbon and anchor impellers. Based on mixing time with respect to mixing energy level, three impeller types (ribbon, CF-2, and MHS 3/39°) stand out when agitating highly viscous fluids, of these mixing with two MHS 3/39° impellers requires the lowest power input to homogenize the manure slurry. A comparison of digestion material demonstrates that the mixing energy varies with manure type and total solids concentration to obtain a given mixing time. Moreover, an in-depth discussion about the CFD strategy, the influences of flow regime and impeller type on mixing characteristics, and the intrinsic relation between mixing and flow field is included.     

Selective suppression of bacterial contaminants by process conditions during lignocellulose based yeast fermentations

Background

When scaling up lignocellulose-based ethanol production, the desire to increase the final ethanol titer after fermentation can introduce problems. A high concentration of water-insoluble solids (WIS) is needed in the enzymatic hydrolysis step, resulting in increased viscosity, which can cause mass and heat transfer problems because of poor mixing of the material. In the present study, the effects of mixing on the enzymatic hydrolysis of steam-pretreated spruce were investigated using a stirred tank reactor operated with different impeller speeds and enzyme loadings. In addition, the results were related to the power input needed to operate the impeller at different speeds, taking into account the changes in rheology throughout the process.

Results

A marked difference in hydrolysis rate at different impeller speeds was found. For example, the conversion was twice as high after 48 hours at 500 rpm compared with 25 rpm. This difference remained throughout the 96 hours of hydrolysis. Substantial amounts of energy were required to achieve only minor increases in conversion during the later stages of the process.

Conclusions

Impeller speed strongly affected both the hydrolysis rate of the pretreated spruce and needed power input. Similar conversions could be obtained at different energy input by altering the mixing (that is, energy input), enzyme load and residence time, an important issue to consider when designing large-scale plants.     

Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection

The aim of this work was to select an efficient impeller to be used in a stirred reactor for the enzymatic hydrolysis of sugar cane bagasse. All experiments utilized 100 g (dry weight)/l of steam-pretreated bagasse, which is utilized in Brazil for cattle feed. The process was studied with respect to the rheological behavior of the biomass hydrolysate and the enzymatic conversion of the bagasse polysaccharides. These parameters were applied to model the power required for an impeller to operate at pilot scale (100 l) using empirical correlations according to Nagata [16]. Hydrolysis experiments were carried out using a blend of cellulases, β-glucosidase, and xylanases produced in our laboratory by Trichoderma reesei RUT C30 and Aspergillus awamori. Hydrolyses were performed with an enzyme load of 10 FPU/g (dry weight) of bagasse over 36 h with periodic sampling for the measurement of viscosity and the concentration of glucose and reducing sugars. The mixture presented pseudoplastic behavior. This rheological model allowed for a performance comparison to be made between flat-blade disk (Rushton turbine) and pitched-blade (45°) impellers. The simulation showed that the pitched blade consumed tenfold less energy than the flat-blade disk turbine. The resulting sugar syrups contained 22 g/l of glucose, which corresponded to 45% cellulose conversion.     

Mixing analysis of PCS slurries in a horizontal scraped surface bioreactor

Horizontal rotating reactors offer many advantages for enzymatic hydrolysis of viscous biomass slurries; however, they do not provide homogenous mixtures since motion is only in the angular direction. Multi-directional mixing is important for dispersing enzymes and carrying products away from reaction sites. The objective here was to experimentally quantify mixing times and axial dispersion coefficients in a horizontal rotating bioreactor. Mixing times were of the same order as reaction times, indicating that enzymatic hydrolysis could be as much controlled by diffusion and mixing effects as by the complex reaction mechanism. The dispersion coefficient for the highest solids slurry was 20× less than the lowest solids slurry, which is indicative of the difference in free water and the magnitude change of viscosity with relatively small addition of solids. The slow mixing times and low dispersion may be an acceptable tradeoff with significantly lower power requirements compared to a conventional vertical reactor.     

Triarylmethane Dye Decolorization by Pellets of Pycnoporus sanguineus: Statistical Optimization and Effects of Novel Impeller Geometry

A study was carried out to optimize selected parameters for decolorization of a triarylmethane dye, such as crystal violet by white rot fungus, Pycnoporus sanguineus, pellets. The parameters studied were initial dye concentration (ppm), agitation speed (rpm), and process time (days) and were optimized using response surface methodology (RSM). It is shown that process time, agitation speed, and their interactions have significant effects on the decolorization process. Following the optimization, the decolorization study was extended to a stirred tank reactor (STR) process. Effects of different geometry of impellers on the decolorization process and power consumption were studied. Novel impeller geometries, such as 180° curved blade and 60° angled blade impellers, were used in the STR. The application of 180° curved blade impeller resulted in higher percentage of decolorization at a relatively less power consumption as compared with 60° angled blade impeller.     

Reactor properties of a high-speed bead mill for microbial cell rupture

Laboratory and pilot-plant high-speed bead mills of 0.6 and 5 liter capacity and consisting of four and five impellers in series, respectively, were used to follow the batch and continuous disruption of bakers' yeast (Saccharomyces cerevisiae). The mills are not scaled equivalents. Throughputs ranging from 1 × 10^?6m³/sec to 12 × 10^?6m³/sec for the 0.6 liter mill and from 16 × 10^?6m³/sec to 100 × 10^?6m³/sec for the 5 liter mill were used for continuous disruption studies. Variables studied included the effect of impeller tip speed, temperature, and packed yeast concentration (ranging from 15 to75% by weight packed yeast). Disruption kinetics, as measured by the release of soluble protein, followed a first-order rate equation, the rate constant being a function of impeller tip speed and yeast concentration. For continuous disruption studies the bead mills behaved as a series of continuous stirred-tank reactors, each impeller forming a reactor. In the smaller mill a considerable degree of backflow between the reactors was evident. For certain mixing conditions the maximum amount of releasable protein was dependent on the impeller geometry, construction material, and also the concentration of packed yeast. The relative power efficiencies of the two mills are discussed along with possible criteria for scaling of bead mills.     

Bioconversion of waste cellulose by using an attrition bioreactor

A new type of reactor, an attrition bioreactor, was tested to achieve a higher rate and extent of enzymatic saccharification of cellulose than is possible with conventional methods. The reactor consisted of a jacketted stainless-steel vessel with shaft, stirrer, and milling media, which combined the effect of the mechanical action of wet milling with cellulose hydrolysis. The substrates tested were newsprint and white-pine heartwood. The performance of the reactor was excellent. The extent and rate of enzymatic hydrolysis could be markedly improved over other methods. The power consumption of the attrition bioreactor was also measured. The cellulase enzyme deactivation during attrition milling was not significant.     

Effect of Impeller Clearance on Flow Structure and Mixing in Bioreactor with Two‐Stage Impellers

The effect of impeller clearance on flow structure and mixing time was simulated using a commercial software package CFX 4.3 and was measured experimentally. The mixing time calculated by simulation exhibited good agreement with the experimental data. The trend of forming independent flow compartments by each impeller became stronger as the clearance between two impellers increased. The homogeneity in the bioreactor was affected mainly by flow exchange between the compartments by each impeller. The most efficient mixing occurred when the impeller clearance was in the range of 0.2–0.4 vessel diameter.     

螺带桨搅拌在木质纤维素酸预处理反应器中的应用简

在干式稀酸预处理的反应器中采用螺带桨搅拌器,对秸秆预处理体系进行混合。在带有螺带式搅拌的预处理过程中,在质量分数2.0％和2.5％的H2 SO4用量条件下,预处理后72 h秸秆的酶解糖化得率分别为77.55％和87.11％,比静态预处理得到的得率分别增长了7.6％和2.4％,抑制物的生成显著降低。通过计算流体力学方法验证,螺带桨搅拌器可以有效地改善玉米秸秆在稀酸预处理过程中的蒸汽和秸秆两相的混合情况。     

Performance intensification of a stirred bioreactor for fermentative biohydrogen production

In this study, the biohydrogen (bioH₂) production of a microbial consortium was optimized by adjusting the type and configuration of two impellers, the mixing regimen and the mass transfer process (K_La coefficients). A continuous stirred-tank reactor (CSTR) system, with a nonstandard geometry, was characterized. Two different mixing configurations with either predominant axial (PB4 impeller) or radial pumping (Rushton impeller) were assessed and four different impeller configurations to produce bioH₂. The best configuration for an adequate mixing time was determined by an ANOVA analysis. A response surface methodology was also used to fully elucidate the optimal configuration. When the PB4 impellers were placed in best configuration, c/Dt?=?0.5, s/Di?=?1, the maximum bioH₂ productivity obtained was 440?mL?L^?1?hr^?1, with a bioH₂ molar yield of 1.8. The second best configuration obtained with the PB4 impellers presented a bioH₂ productivity of 407.94?mL?L^?1?hr^?1. The configurations based on Rushton impellers showed a lower bioH₂ productivity and bioH₂ molar yield of 177.065?mL?L^?1?hr^?1 and 0.71, respectively. The experiments with axial impellers (PB4) showed the lowest K_La coefficient and the highest bioH₂ production, suggesting that mixing is more important than K_La for the enhanced production of bioH_2.     

Mixing studies in bioreactors

Blend times and power consumptions were determined for different arrangements of two equal diameter impellers, a high efficiency A310 and a “Dumbo Ear” impeller with three large, “elephant ear” blades designed for low shear agitation. A 9 l round-bottomed, unbaffled bioreactor was used in these studies. Blend times were taken as the time for the disappearance of the pink color of a basic solution of phenolphthalein on neutralization by excess acid, and the power consumption was obtained from torque measurements. The mixing results show that the Dumbo Ear impeller gives shorter blend times than the A310?at equal rotational speeds for most of the conditions studied. As expected, the Dumbo Ear impeller consumes more power than the A310?at the same rotational speed, due to its large area blades. However, the Dumbo Ear impeller also gives shorter blend times than the A310?at equal power consumptions.     

Process development of starch hydrolysis using mixing characteristics of Taylor vortices

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor–Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.     

Mathematical modeling of non-ideal mixing continuous flow reactors for anaerobic digestion of cattle manure

Most conventional digesters used for animal wastewater treatment include continuously stirred-tank reactors. While imperfect mixing patterns are more common than ideal ones in real reactors, anaerobic digestion models often assume complete mixing conditions. Therefore, their applicability appears to be limited. In this study, a mathematical model for anaerobic digestion of cattle manure was developed to describe the dynamic behavior of non-ideal mixing continuous flow reactors. The microbial kinetic model includes an enzymatic hydrolysis step and four microbial growth steps, together with the effects of substrate inhibition, pH and thermodynamic considerations. The biokinetic expressions were linked to a simple two-region liquid mixing model, which considered the reactor volume in two separate sections, the flow-through and the retention regions. Deviations from an ideal completely mixed regime were represented by changing the relative volume of the flow-through region (a) and the ratio of the internal exchange flow rate to the feed flow rate (b). The effects of the hydraulic retention time, the composition of feed, the initial conditions of the reactor and the degree of mixing on process performance can be evaluated by the dynamic model. The simulation results under different conditions showed that deviations from the ideal mixing regime decreased the methane yield and resulted in a reduced performance of the anaerobic reactors. The evaluation of the impact of the characteristic mixing parameters (a) and (b) on the anaerobic digestion of cattle manure showed that both liquid mixing parameters had significant effects on reactor performance.     

Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis: Part II

Lignocellulose breakdown in biorefineries is facilitated by enzymes and physical forces. Enzymes degrade and solubilize accessible lignocellulosic polymers, primarily on fiber surfaces, and make fibers physically weaker. Meanwhile physical forces acting during mechanical agitation induce tearing and cause rupture and attrition of the fibers, leading to liquefaction, that is, a less viscous hydrolysate that can be further processed in industrial settings. This study aims at understanding how mechanical agitation during enzymatic saccharification can be used to promote fiber attrition. The effects of reaction conditions, such as substrate and enzyme concentration on fiber attrition rate and hydrolysis yield were investigated. To gain insight into the fiber attrition mechanism, enzymatic hydrolysis was compared to hydrolysis by use of hydrochloric acid. Results show that fiber attrition depends on several factors concerning reactor design and operation including drum diameter, rotational speed, mixing schedule, and concentrations of fibers and enzymes. Surprisingly, different fiber attrition patterns during enzymatic and acid hydrolysis were found for similar mixing schedules. Specifically, for tumbling mixing, slow continuous mixing appears to function better than faster, intermittent mixing even for the same total number of drum revolutions. The findings indicate that reactor design and operation as well as hydrolysis conditions are key to process optimization and that detailed insights are needed to obtain fast liquefaction without sacrificing saccharification yields.     

Strategies for penicillin fermentation in tower-loop reactors

Since it has not been possible to produce penicillin in tower-loop reactors with highly viscous filamentous molds of Penicillium chrysogenum which are employed in stirred-tank reactors, a new strategy has been developed to avoid the formation of this morphology and to use the pellet form of the fungi. When employing definite impeller speeds in the subculture in connection with definite inoculum amounts and substrate concentrations in the main culture (bubble column), it is possible to generate a suspension of isolated small pellets, which shows a low broth viscosity up to a sediment content of 45% over the entire fermentation time. Volumetric mass-transfer coefficients k(L)as are by a factor of 4 to 5 higher in these pellet suspensions than in filamentous broths. It was easy to maintain the necessary oxygen supply for penicillin production in these pellet suspensions. Under these conditions the specific penicillin productivities were higher with regard to power input (up to 90%), biomass, and consumed substrate than in the stirred-tank reactors with highly viscous filamentous morphology of the fungi. Under nonoptimized operating conditions the absolute penicillin production in the tower loop was 35% lower than in the stirred-tank reactor due to lower possible biomass concentrations. The separation of the biomass, and therefore the penicillin recovery, is much simpler when employing pellets. It is shown how the particular mass transfer resistances at the gas/liquid and liquid/pellet interfaces and within the pellets change with the pellet diameter. There should be a particular pellet diameter at which penicillin productivity has its maximum. These investigations indicate that the use of tower-loop reactors can, in the future, be an alternative for more economical penicillin production methods.     

Studies with a multiple-rod mixing impeller

The performance of a multiple-rod mixing impeller was compared to that of conventional turbine impellers in viscous novobiocin beers. The advantages of the multiple-rod impeller were found to be: (1) the power requirement was independent of changes in apparent viscosity of the fermentation beer; and (2) it gave the same novobiocin yield and oxygen-availability rate at about one-half of the power required by turbines.