Studies of the mixing character and flow distribution in mycelial fermentation broths

The influence of two mixing systems on the principal parameters of mycelial fermentations of Aspergillus niger, Fusicoccum amygdali Del. and Fusarium moniliforme Sheld. as well as their metabolite citric acid, fusicoccin and gibberellic acid production was analyzed from the viewpoint of flow energy distribution in a bioreactor. The growth and metabolite synthesis during fermentation was compared under different mixing conditions in the fermenter FU-8 with a turbine mixing system (TMS) and a counterflow mixing system (CMS). It was found that the growth, productivity and respiration characteristics as well as the morphology of these cultures varied dependent on the mixing system and agitation regime used. The counterflow mixing system was more favourable for large agglomerates (F. amygdali) or soft pellets (A. niger) forming fungi, while the turbine mixing system was more effective for F. moniliforme growing in the form of small clumps and freely dispersed hyphae. Flow characteristics under different mixing conditions were analyzed in a model fermenter. The kinetic energy of flow fluctuations was measured in gassed and ungassed water and different fermentation broth systems by using a Stirring Intensity Measuring Device (SIMD-F1). The difference of the energy values at different points was better expressed in the fermenter with a turbine mixing system in comparison with that having a counterflow mixing system. High viscous F. amygdali and A. niger broth provided higher energy values compared to water and low viscous F. moniliforme broth. It was observed that the intensity of growth and the intensity of the synthesis decreased at very high energy values, which was obviously connected to the influence of the irreversible shear stress on the mycelial morphology.     

Studies on transfer processes in mixing vessels: effect of gas on solid-liquid hydrodynamics using modified Rushton turbine agitators

The effect of gas on solid-liquid hydrodynamics in mixing vessels was studied to determine the agitation speed required to just completely suspend all the particles under gassed conditions, N _jsg and by measurement of the power consumption associated with this agitation speed. The solid particles have a mean diameter between 15–1000 μm. For their mixing are used standard and modified Rushton turbine agitators positioned singly or doubly on the same shaft. The modified turbine with a surface fraction of the perforations equal to 0.353 (TP3) was obtained through increase in the blade height of the Rushton turbine simultaneously with the perforation of the blade surface. The filled surface of the modified blade is equal to the blade surface of the standard Rushton turbine.     

Hydrodynamic and kinetic study of cellulase production by Trichoderma reesei with pellet morphology

Numerical simulations and experimental validation were performed to understand the effects of hydrodynamics on pellet formation and cellulase production by filamentous T. reesei. The constructed model combined a steady-state multiple reference frame (MRF) approach describing mechanical mixing, oxygen mass transfer, and non-Newtonian flow field with a transient sliding mesh approach and kinetics of oxygen consumption, pellet formation, and enzyme production. The model was experimentally validated at various agitation speeds in a two-impeller Rushton turbine fermentor. Results from simulation and experimentation showed that higher agitation speeds led to increases in the pellet diameter and the proportion of pelletized (vs. filamentous) forms of the biomass. It also led to increase in dissolved oxygen mass transfer rate in shear-thinning fluid and cellulase productivity. The extent of these increases varied considerably among agitation speeds. Pellet formation and morphology were presumably affected within a viscosity-dependent shear-rate range. Cellulase activity and cell viability were shown to be sensitive to impeller shear. A maximum cellulase activity of 3.5 IU/mL was obtained at 400 rpm, representing a twofold increase over that at 100 rpm.     

Impeller types and feeding modes influence the morphology and protein expression in the submerged culture of<Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The influences of impeller types on morphology and protein expression were investigated in a submerged culture ofAspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production for both the intracellular heterologous protein (β-glucuronidase) and the extracellularly homologous protein (α-amylase). The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation ofA. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged fed-batch fermentation of recombinantA. oryzae.     

Improvement of foam breaking and oxygen-transfer performance in a stirred-tank fermenter

This study examined a stirred-tank fermenter (STF) containing low-viscosity foaming liquids with an agitation impeller and foam-breaking impeller mounted on the same shaft. Results showed that the performance of the foam-breaking impeller can be improved by changing a conventional six-blade turbine impeller into a rod impeller as the agitation impeller. The volumetric oxygen-transfer coefficient, k _L a, in the mechanical foam-control method (MFM) using a six-blade vaned disk as the foam-breaking impeller in the STF with the rod impeller was approximately five times greater than that of the chemical foam-control method (CFM) adding an anti-foaming agent in the STF with the six-blade turbine impeller. Application of the present method to the cultivation of Saccharomyces cerevisiae K-7 demonstrated that the cultivation time up to the maximum cell concentration was remarkably shorter than that achieved using a conventional CFM.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter‐ion and iso‐propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04‐fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:845–855, 2014     

Determination of a scale-up factor from mixing time studies in orbitally shaken bioreactors

Efficient mixing in bioreactors is essential in order to avoid concentration gradients which can be harmful for mammalian cells. To study mixing and its scalability in orbitally shaken cylindrical bioreactors, we measured mixing times in containers with nominal volumes from 2 to 1500 L with a colorimetric method using two pH indicators. Four operating parameters were tested: the liquid height, the shaking diameter, the agitation rate, and the inner diameter of the container. The mixing time decreased as the agitation rate increased until a minimal value was reached. As the shaking diameter was reduced, a higher agitation rate was needed to reach the minimal mixing time. The liquid height did not have a significant effect on the mixing time, but for a constant volume, an increase of the inner diameter slightly reduced the mixing time. The fastest mixed zones were close to the wall of the container while the zone in the center of the bulk liquid was the last to achieve homogeneity. Our study showed that the free-surface shape correlated with the mixing regime and that by keeping the inner-to-shaking diameter ratio as well as the Froude number (Fr) constant, the free-surface shapes and the mixing regimes of a 1500-L bioreactor could be mimicked in a 30-L bioreactor. We concluded that the mixing in orbitally shaken cylindrical bioreactors ensures homogeneity for mammalian cell cultures at scales up to 1500 L and that the inner-to-shaking diameter is a suitable scale-up factor for mixing.     

Effect of aeration and agitation on synthesis of poly (γ-glutamic acid) in batch cultures of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324

The effects of aeration and agitation on the production and molecular weight of poly (γ-glutamic acid) (PGA) were systematically investigated in batch fermentor cultures of Bacillus licheniformis NCIM 2324. A high aeration rate and agitation speed enhanced the growth of B. licheniformis NCIM 2324, but did not always lead to high PGA production. Additionally, PGA production actually decreased at very high aeration rates and agitation speeds. The maximum PGA concentration was obtained at 750 rpm and 1 vvm. Rheological studies revealed that fermentation broth during production of PGA exhibited pseudoplastic behavior. The effects of aeration and agitation on the molecular weight of PGA were also studied, and the rate and extent of the decrease in the molecular weight of PGA as a function of time were found to be much greater at high aeration than low aeration. The PGA production of 46.34 g/L with a specific productivity of 0.17 g-PGA/g-biomass/ h and a PGA yield of 0.48 with respect to total substrate observed in the present study are much higher than the values reported in previously conducted studies.     

Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Studies on the production of bacterial rennet in a pilot plant fermentor

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.     

Studies supporting the use of mechanical mixing in large scale beer fermentations

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO₂ evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer’s yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO₂ evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing.     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Production of chum salmon cystatin from the recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> optimized using response surface methodology

Chum salmon cystatin was overexpressed on Saccharomyces cerevisiae YPH 499. At first, the culture condition for the production of recombinant chum salmon cystatin (RC) by S. cerevisiae YPH 499 was optimized in a shake flask using response surface methodology. Three independent variables; medium pH, inducing time, and the amount of inducing assistant, were analyzed to get the optimal condition for the production of RC. The results were fitted to a second-order polynomial equation, in which the determination coefficient (R ²) was 0.904. The highest RC production in a shake flask, 0.57 U/mL was obtained at 5.7 of medium pH, 6.7 h of inducing time, and 5.6 g/L of inducing assistant. Based on the results of shake flask, the effects of agitation and aeration rates on the production of RC by S. cerevisiae YPH 499 were determined for scaleup in a fermentor. The highest production of RC in a fermentor, 0.56 U/mL was obtained at 350 rpm of agitation rate and 1.0 vvm of aeration rate. RC at 100 μg/g showed the highest inhibitory activity against the autolysis of Alaska pollock surimi based on the analysis of TCA-soluble peptides.     

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。     

Fluid mixing and oxygen transfer in cell suspensions ofTaxus chinensis in a novel stirred bioreactor

In high-density plant cell cultures, mixing and mass transfer are two key issues, which should be emphasized for process optimization. In this work, both mixing and oxygen transfer characteristics of cell suspensions ofTaxus chinensis were studied in a new centrifugal impeller bioreactor with a working volume of 1.2 L. The mixing time (t _M) and the volumetric oxygen transfer coefficient (K _L a) under different operational conditions were determined in both tap water and cell suspensions of 100–400 g fresh weight/L (i.e., 5.65–23.1 g DW/L). At an aeration rate of 0.1 L/min,t _M decreased from 10.6s at 30 rpm to 2.89 s at 200 rpm under 100 g FW/L, and from 9.63 s (120 rpm) to 4.05 s (300 rpm) under 400 g FW/L. Compared with the effect of agitation, aeration was less significant to the suspension mixing. At a relatively high agitation speed (e.g., 200 rpm),t _M remained almost the same even though aeration rate was changed from 0.1 to 0.4 L/min. Thet _M value increased slowly from 3.98 to 5.26 s at 120 rpm when the cell density was raised from 100 to 250 g FW/L. A rapid increase of botht _M and the suspension viscosity was observed at a cell density above 300 g FW/L. As expected, theK _L a value increased with an increase of aeration rate and agitation speed, but decreased with an increase of cell density. The quantitative data obtained here are useful to investigate the effect of mixing stress on the cell physiology and metabolism ofTaxus chinensis in the bioreactor. This paper is dedicated by JJZ to his colleague Prof. Jun-Tang Yu on the occasion of his 70 birthday.     

Dissolved oxygen measurements in pilot- and production-scale novobiocin fermentations

Dissolved oxygen measurements were made in pilot (20 and 250 l.) and production scale (15,000 l.) novobiocin fermentations. Bulk mixing was found to be incomplete in pilot tanks with turbine impellers of D/T = 0.40 (where D is impeller diameter, and T is tank diameter) but appeared homogeneous with impellers of D/T = 0.69. In the former case, the respiration rate was presumably limited by insufficient oxygen supply in areas of poor bulk mixing, whereas, in the latter case, the major resistance was between the bulk of the liquid and the cell (intraclump resistance). Higher agitator speeds decreased the gas–liquid resistance proportionally more than they reduced the liquid-cell resistance. In production fermentors, dissolved oxygen measurements indicated that bulk mixing was complete with each of the three impeller sizes tested (D/T = 0.28, 0.33, and 0.43), but that the respiration rate was again limited, mainly by a resistance between the bulk of the liquid and the cell.     

Mixing state of xanthan gum solution in an aerated and agitated fermentor—Effects of impeller size on volumes of mixing regions and circulation time distribution

The effects of the impeller diameter and width on the volumes of the micromixing and macromixing regions, and on the circulation time distribution were investigated at various agitation speeds to formulate the relationships of them in emperical equations. A fermentor was a 10-l capacity, which was equipped with a turbine impeller with six flat balades and aerated at 1 vvm. It was found that the volumes of the micromixing and macromixing regions depended on the tip speed of the impeller, ND, and the discharging performance of the impeller, ND²W, respectively, in the xabthan gum solution with concentrations of 0.9, 1.8, 2.7, and 3.9%. Empirical equations were derived to estimate the volume of each mixing region from the impeller diameter, D, impeller width, W, agitation speed, N, and consistency coefficient of the xanthan gum solution. On the other hand, the circulation time distribution could be estimated empirically from only the impeller diameter and agitation speed, regardless of variation in the impeller width and consistency coefficient of the xanthan gum solution tested.