Scale up of a viscous fungal fermentation: application of scale-up criteria with regime analysis and operating boundary conditions

The scale up of the novel, pharmaceutically important pneumocandin (B(0)), from the filamentous fungus Glarea lozoyensis was successfully completed from pilot scale (0.07, 0.8, and 19 m(3)) to production scale (57 m(3)). This was accomplished, despite dissimilar reactor geometry, employing a combination of scale-up criteria, process sensitivity studies, and regime analysis using characteristic time constants for both oxygen mass transfer and bulk mixing. Dissolved oxygen tension, separated from the influence of agitation by gas blending at the 0.07 m(3)-scale, had a marked influence on the concentrations of pneumocandin analogs with different levels of hydroxylation, and these concentrations were used as an indicator of bulk mixing upon scale up. The profound impact of dissolved oxygen tension (DOT) (low and high levels) on analog formation dictated the use of constant DOT, at 80% air saturation, as a scale-up criterion. As a result k(L)a, Oxygen uptake rate (OUR) and hence the OTR were held constant, which were effectively conserved across the scales, while the use of other criterion such as P(g)/V(L), or mixing time were less effective. Production scale (57 m(3)) mixing times were found to be faster than those at 19 m(3) due to a difference in liquid height/tank diameter ratio (H(L)/D(T)). Regime analysis at 19 and 57 m(3) for bulk mixing (t(c)) and oxygen transfer (1/k(L)a) showed that oxygen transfer was the rate-limiting step for this highly shear thinning fermentation, providing additional support for the choice of scale-up criterion.     

丝状真菌Glarea lozoyensis SIIA-F1108产纽莫康定B0发酵条件的研究

【目的】采用响应面法优化丝状真菌Glarea lozoyensis SIIA-F1108发酵生产纽莫康定B_0培养基,提高发酵产量;通过氮源优化,降低发酵液菌体浓度,改善发酵过程的溶氧水平。【方法】采用Plackett-Burman设计和响应面法进行培养基优化,筛选出对纽莫康定B_0产量具有显著影响的因素;通过最陡爬坡实验及Box-Behnken设计,并利用Design-Expert软件对实验数据进行回归分析,得到优化的发酵培养基配方;通过对优化培养基中氮源组分进行全因子实验,最终得到高产量和低菌体浓度发酵培养基。【结果】实验数据表明:甘露醇、脯氨酸和葡萄糖对纽莫康定B_0产量影响最大;最佳浓度分别为甘露醇167.3 g/L、脯氨酸26.1 g/L、葡萄糖28.5 g/L。采用优化后的培养基进行摇瓶发酵,纽莫康定B_0产量达到了1 840 mg/L,较优化前提高了42%,与预测结果一致。用硫酸铵部分替换棉籽饼粉后,发酵液菌体浓度降低,在100 L发酵罐上对优化后的结果做了进一步的验证,纽莫康定B_0产量达到1 980 mg/L。【结论】模型预测值与实验值有较高吻合度,具备较高可信度和显著性,发酵产量提高了42%,响应面实验设计和分析方法能够有效地用于丝状真菌Glarea lozoyensis SIIA-F1108产纽莫康定B_0发酵培养基进行优化。通过调整培养基中的氮源组成,降低了发酵液菌体浓度,改善了发酵过程的溶氧水平。     

Influence of the intensity of oxygen mass transfer on the biosynthesis of amphotericin B

Influence of oxygen mass transfer intensity characterized by the rate of oxygen dissolution (S) and the agitation rate (n), as well as influence of dissolved oxygen concentration on the process of amphotericin B biosynthesis was studied. It was shown that S = 40 and 110 mg/l. min and n = 450 and 800 min-1 were respectively the lower and the upper levels of the optimal conditions by oxygen mass transfer during amphotericin B biosynthesis. When biosynthesis of amphotericin B was conducted under conditions of the optimal oxygen mass transfer, the dissolved oxygen concentration of about 12 to 15 per cent of the saturation level was critical for the culture respiration. Inhibition of the culture respiration and antibiotic synthesis was induced under conditions of increased oxygen mass transfer intensity (S greater than 110 mg/l. min and n greater than 800 min-1) by high intensity mechanical agitation of the fermentation broth. Under conditions of decreased oxygen mass transfer (S less than 40 mg/l. min and n = less than 450 min-1) it was induced by insufficient supply of oxygen to the culture. On the basis of the results it was shown possible to control the aeration and agitation conditions by the rate of oxygen uptake and dissolved oxygen concentration. The data should be considered in optimization of aeration and agitation conditions in biosynthesis of amphotericin B in large fermenters.     

Effect of oxygen concentration on the growth of Nannochloropsis sp. at low light intensity

In large-scale microalgal production in tubular photobioreactors, the build-up of O(2) along the tubes is one of the major bottlenecks to obtain high productivities. Oxygen inhibits the growth, since it competes with carbon dioxide for the Rubisco enzyme involved in the CO(2) fixation to generate biomass. The effect of oxygen on growth of Nannochloropsis sp. was experimentally determined in a fully controlled flat-panel photobioreactor operated in turbidostat mode using an incident photon flux density of 100?μmol photons m(-2) s(-1) and with only the oxygen concentration as variable parameter. The dissolved oxygen concentration was varied from 20 to 250% air saturation. Results showed that there was no clear effect of oxygen concentration on specific growth rate (mean of 0.48?±?0.40?day(-1)) upon increasing the oxygen concentration from 20% to 75% air saturation. Upon further increasing the oxygen concentration, however, a linear decrease in specific growth rate was observed, ranging from 0.48?±?0.40?day(-1) at a dissolved oxygen concentration of 75% air saturation to 0.18?±?0.01?day(-1) at 250% air saturation. In vitro data on isolated Rubisco were used to predict the quantum yield at different oxygen concentrations in the medium. The predicted decrease in quantum yield matches well with the observed decrease that was measured in vivo. These results indicate that the effect of oxygen on growth of Nannochloropsis sp. at low light intensity is only due to competitive inhibition of the Rubisco enzyme. At these sub-saturating light conditions, the presence of high concentrations of oxygen in the medium induced slightly higher carotenoid content, but the increased levels of this protective antioxidant did not diminish the growth-inhibiting effects of oxygen on the Rubisco.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Vancomycin production is enhanced in chemostat culture with biomass-recycle

Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited chemostat cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D= 0. 14 h-1). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of chemostat culture without biomass-recycle. The specific production rate (qvancomycin) was maximal when the biomass-recycle ratio (R) was 0.13 (D= 0.087 h-1). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44 (D= 0.056 h -1) compared to chemostat culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to chemostat culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics.     

The influence of fish size size on the avoidance of hypoxia and oxygen selection by largemouth bass

When an oxygen gradient ranging from c . 10 to 95% air saturation was formed in a 5 m chamber, largemouth bass Micropterus salmoides avoided water in which dissolved oxygen values were <27% air saturation. There was a significant ( P <0·05) correlation between fish mass and the level of dissolved aquatic oxygen that was selected. Small fish (23–500 g) utilized waters of lower oxygen levels than did the larger fish (1000–3000 g). The results of this study suggest that largemouth bass are able to sense and avoid hypoxic water, and select aquatic oxygen tensions that maintain their metabolic scope for growth and activity.     

Normal phase high-performance liquid chromatography of pneumocandins: in situ modification of silica with L-proline to separate structural analogues

Lipopeptides such as pneumocandin B(0) are often produced by fermentation processes. Many compounds with similar structures (structural analogues), and hence similar physiochemical properties, are coproduced in the fermentation. We employed high performance liquid chromatography using silica gel as the stationary phase and a ternary ethyl acetate/MeOH/water mobile phase to separate pneumocandin B(0) from these structural analogues. Despite extensive efforts to optimize this system, two key structural analogues, pneumocandin E(0) and pneumocandin B(5), continued to be poorly resolved from the main product peak (pneumocandin B(0)). As a result, feed load was restricted and productivity was limited. In situ modification of the silica gel stationary phase with l-proline or other amino acids significantly enhances the resolution of the two key structural analogues from the compound of interest, enabling a two-fold increase in productivity. Results of a systematic study showed that the amine group in l-proline and other amino acids plays a key role in the modification of the surface of the silica gel to mediate the selectivity enhancement.     

Experimental evaluation of liquid film resistance in oxygen transport to microbial cells

A membrane probe was used to monitor the dissolved oxygen concentrations in continuous cultures of Candida utilis and Micrococcus roseus growing at low dissolved oxygen concentrations and various agitation levels. For the yeast fermentations, increasing the agitation level within the range of 0.1 to 0.3 w per liter lowered steady-state dissolved oxygen concentrations in the fermentor. The steady-state dissolved oxygen concentration in the fermentor was not influenced by the agitation level within the range of 0.3 to 1.8 w per liter. With M. roseus, no effect of agitation on steady-state dissolved oxygen concentrations in the fermentor was observed within the range of 0.1 to 1.8 w per liter. It was concluded that, under the conditions used, a measurable transfer barrier from the liquid to the yeast cells existed at agitation levels below 0.3 w per liter and that this barrier did not exist at agitation levels above 0.3 w per liter. The transfer barrier from the liquid to the yeast surface could be represented by a stagnant film of liquid 0.6 × 10^-4 cm thick surrounding the cell at an agitation level of 0.10 w per liter. This film represented an oxygen concentration drop of 1.3 × 10^-7 M from the bulk of the medium to the cells under the experimental conditions.     

Tubular photobioreactor design for algal cultures.

Principles of fluid mechanics, gas-liquid mass transfer, and irradiance controlled algal growth are integrated into a method for designing tubular photobioreactors in which the culture is circulated by an airlift pump. A 0.2 m(3) photobioreactor designed using the proposed approach was proved in continuous outdoor culture of the microalga Phaeodactylum tricornutum. The culture performance was assessed under various conditions of irradiance, dilution rates and liquid velocities through the tubular solar collector. A biomass productivity of 1.90 g l(-1) d(-1) (or 32 g m(-2) d(-1)) could be obtained at a dilution rate of 0.04 h(-1). Photoinhibition was observed during hours of peak irradiance; the photosynthetic activity of the cells recovered a few hours later. Linear liquid velocities of 0.50 and 0.35 m s(-1) in the solar collector gave similar biomass productivities, but the culture collapsed at lower velocities. The effect of dissolved oxygen concentration on productivity was quantified in indoor conditions; dissolved oxygen levels higher or lower than air saturation values reduced productivity. Under outdoor conditions, for given levels of oxygen supersaturation, the productivity decline was greater outdoors than indoors, suggesting that under intense outdoor illumination photooxidation contributed to loss of productivity in comparison with productivity loss due to oxygen inhibition alone. Dissolved oxygen values at the outlet of solar collector tube were up to 400% of air saturation.     

The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

Agitator speed and dissolved oxygen effects in xanthan fermentations

Agitation speed affects both the extent of motion in Xanthan fermentation broths because of their rheological complexity and the rate of oxygen transfer. The combination of these two effects causes the dissolved oxygen concentration and its spatial uniformity also to change with agitator speed. Separating these complex interactions has been achieved in this study in the following way. First, the influence of agitation speeds of 500 and 1000 rpm has been investigated at a constant nonlimiting dissolved oxygen concentration of 20% of air saturation using gas blending. Under these controlled dissolved oxygen conditions, the results demonstrate that the biological performance of the culture was independent of agitation speed as long as broth homogeneity could be ensured. With the development of increasing rheological complexity lending to stagnant regions at Xanthan concentrations >20 g/L, it is shown that the superior bulk mixing achieved at 1000 rpm, compared with 500 rpm, leading to an increased proportion of the cells in the fermentor to be metabolically active and hence higher microbial oxygen uptake rates, was responsible for the enhanced performance. Second, the effects of varying dissolved oxygen are compared with a control in each case with an agitator speed of 1000 rpm to ensure full motion, but with a fixed, nonlimiting dissolved oxygen of 20% air saturation. The specific oxygen uptake rate of the culture in the exponential phase, determined using steady-state gas analysis data, was found to be independent of dissolved oxygen above 6% air saturation, whereas the specific growth rate of the culture was not influenced by dissolved oxygen, even at levels as low as 3%, although a decrease in Xanthan production rate could be measured. In the production phase, the critical oxygen level was determined to be 6% to 10%, so that, below this value, both specific Xanthan production rate as well as specific oxygen uptake rate decreased significantly. In addition, it is shown that the dynamic method of oxygen uptake determination is unsuitable even for moderately viscous Xanthan broths. Copyright 1998 John Wiley & Sons, Inc.     

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and k _L a). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.List of Symbols DO % sat. Dissolved oxygen concentration - k _L a h^–1 Gas-liquid mass transfer coefficient - OUR mmol/dm³h Oxygen uptake rate - P/V KW/m³ Agitator power per unit volume - V _s m/s Superficial air velocity - _app cP Apparent viscosity     

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation

The role of dissolved oxygen (DO) and function of agitation in hyaluronic acid fermentation by Streptococcus zooepidemicus ATCC 39920 were explored. With controlled DO levels, it was found that the present strain grew as well under both aerobic and anaerobic conditions. Dissolved oxygen plays a role as a stimulant in the HA synthesis; the intrinsic factor affecting the efficiency of HA synthesis is DO level; and there existed a critical DO level of 5% air saturation for the HA synthesis. On the other hand, agitation functions to mix the broth, to enhance oxygen absorption, but not to release HA capsule. In addition, vigorous agitation would lengthen the operation time. It therefore suggests that the relevant criteria for scaling up the fermentor are to maintain DO level above the critical value, and to provide a mild agitation for homogeneity in the fermentor.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Spatial variation of dissolved gas concentrations in a willow vegetation filter treating landfill leachate

Membrane inlet mass spectrometry (MIMS) was used to monitor continuously and simultaneously the concentrations of dissolved gases (O₂, CO₂, CH₄) within the treatment bed of a willow vegetation filter treating leachate at a landfill site in mid Wales. The distribution of dissolved gasses within the bed was shown to be highly heterogeneous at the small spatial scale with considerable variation between vertical profiles measured simultaneously at different locations. In general, aerobic conditions were observed above the water table with reduced levels of oxygen and increasing levels of carbon dioxide and methane below it. Distinct pockets of oxygen (up to 200 μM) were observed in anaerobic zones and pockets of reduced oxygen and elevated carbon dioxide were observed in the aerobic zone. Pockets of methane were observed in some profiles coexisting with up to 200 μM oxygen at 5 cm depth. These observations confirm the hypothesis that micro-sites exists within the soil/root matrix where aerobic organic matter decomposition and anaerobic processes such as methanogenesis can occur in relatively close proximity to each other. We hypothesise that the distribution of dissolved gases is determined by rapid diffusion of air maintaining aerobic conditions above the water table, removal of oxygen by microbial processes creating anaerobic conditions below the water table and the distribution of willow roots in the soil which create local aerobic zones by oxygen release.     

The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum

The influence of mechanical forces resulting from the rotation of (multiple) turbine impellers on the morphology and penicillin production of Penicillium chrysogenum Panlabs P-1 was investigated in batch fermentations using semi-defined media. Experiments were carried out at three different scales of fermentation, 5 dm³,100 dm³ and 1000 dm³ working volume, with the impeller tip speed ranging from 2.5 to 6.3 m/s. Throughout all fermentations, the dissolved oxygen concentration never fell below the critical value for maximum penicillin production. Morphological measurements using image analysis showed that the mean main hyphal length and mean hyphal growth unit increased during the rapid growth period and then decreased to a relatively constant value dependent on the agitation intensity. The specific rate of penicillin production (q _pen)and the average main hyphal length during the linear penicillin production phase were lower at high agitation speed, which promoted more rapid mycelial fragmentation and a higher branching frequency. Comparison of the results from the three scales showed that impeller tip speed is a poor scale up parameter whereas a term based on mycelial circulation through the zone of high energy dissipation fitted the data well.List of Symbols C.E.R. mmol/(dm³h) Carbon dioxide evolution rate - D m Impeller diameter - D.O.T. % air saturation Dissolved oxygen tension - L _e m Mean effective length or main hyphal length - O.U.R. mmol/(dm³h) Oxygen uptake rate - P W Total power dissipation - q _pen units/(mg dry cell weight h) rate Specific penicillin production - R.Q. Respiratory quotient - 1/t _cs^–1 Circulation frequency     

The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system.

The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.