Effect of oxygen transfer on lipase production by Acinetobacter radioresistens

The influence of oxygen on alkaline lipase production by Acinetobacter radioresistens was studied under two operating modes: controlled dissolved oxygen (DO) concentration and controlled aeration rate. Compared with cell growth, the lipase production depended more extensively on oxygen. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate (OTR) rather than DO concentration. Improvements in OTR, either by aeration or agitation, resulted in an increase in lipase yield and/or a reduction in fermentation time. The formation of A. radioresistens lipase could be described by a mixed-growth-associated model, and the enzyme was mainly a growth-associated product. The overall productivity for the lipase, which depended more strongly on agitation than aeration, could be related with kLa. DO concentration could not be employed in this correlation, though it has been useful as a criterion for ensuring no oxygen limitation in an aerobic fermentation.     

The effects of cells on oxygen transfer in bioreactors

Cells may affect oxygen transfer rates by three mechanisms: respiration of cells accumulated at the gas/liquid interface, physical presence of cells as solid particles, and modification of the medium by cells. These effects were studied experimentally in bubble-aerated bioreactors using baker's yeast at different cell concentrations, agitation speeds, aeration rates, and specific oxygen uptake rates. The overall effect of cells was to enhance oxygen transfer rates. The physical presence of cells as solid particles was found to retard oxygen transfer, presumably due to the lower oxygen permeability in the cell layer accumulated near the bubble surfaces. Cell respiration and medium modification, on the other hand, enhanced oxygen transfer rates. The retardation by nonrespiring cells and the enhancement due to cell respiration were found stronger at higher agitation speeds and lower aeration rates employed. This was attributed to the higher interfacial cell accumulation associated with the smaller bubbles produced under these conditions in the systems studied.     

Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-epsilon-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- epsilon-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-epsilon-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO2) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.     

Model analysis of biological oxygen transfer enhancement in surface-aerated bioreactors

A model was developed to evaluate the effects of cells and surfactants on oxygen transfer in surface-aerated bioreactors. The model assumed the presence of serial layers of adsorbed surfactants and microorganisms directly adjacent to the gas-liquid interface due to their surface activities, followed by a stagnant liquid layer to account for the oxygen transfer resistance in the liquid phase. The interfacial surfactant film, although posing as an additional resistance, was found to have negligible effect on the oxygen transfer rate because of its extremely small thickness as compared to the cell monolayer and the stagnant liquid layer. On the other hand, cells affect oxygen transfer by two mechanisms: the biological enhancement due to the respiration of interfacial cells and the physical blocking resulting from the semipermeable nature of cell bodies. Due to the low specific oxygen uptake rates of the sludges, the two mechanisms were found to be of comparable importance in activated-sludge systems; the oxygen transfer enhancement factor, E, varied from about 0.97 to 1.10 depending on the operating conditions. The biological enhancement effect, however, predominated in fermentations of actively growing bacteria. At relatively low agitation speed (e. g., 300 rpm), the value of E could reach about 3 to 5 in fermentations with high cell concentrations. Effects of other operating variables, such as the agitation intensity, the oxygen content in the mixed liquor, and the bulk cell concentration, on biological oxygen transfer enhancement were also studied. (c) 1992 John Wiley & Sons, Inc.     

Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.     

Determination of in vivo oxygen uptake and carbon dioxide evolution rates from off-gas measurements under highly dynamic conditions

In vivo kinetics of Saccharomyces cerevisiae are studied, in a time window of 150 s, by analyzing the response of O(2) and CO(2) in the fermentor off-gas after perturbation of chemostat cultures by metabolite pulses. Here, a new mathematical method is presented for the estimation of the in vivo oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) directly from the off-gas data in such perturbation experiments. The mathematical construction allows effective elimination of delay and distortion in the off-gas measurement signal under highly dynamic conditions. A black box model for the fermentor off-gas system is first obtained by system identification, followed by the construction of an optimal linear filter, based on the identified off-gas model. The method is applied to glucose and ethanol pulses performed on chemostat cultures of S. cerevisiae. The estimated OUR is shown to be consistent with the independent dissolved oxygen measurement. The estimated in vivo OUR and CER provide valuable insights into the complex dynamic behavior of yeast and are essential for the establishment and validation of in vivo kinetic models of primary metabolism.     

溶氧对变溶菌素发酵的影响

变溶菌素是由球孢链霉菌产生的一种胞外溶菌酶群。它包括几种不同类型的溶菌酶,有着广阔的用途和良好的应用前景。许多研究结果[1,2]表明,它比卵清溶菌酶有更为广泛的溶菌谱,应用范围更广,尤其是在预防和治疗龋齿[3]方面有其独特的优点。在医药上可用作灭菌剂,也可用其作?..     

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.     

Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter

The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.     

Studies on Oxygen Transfer in Submerged Fermentations

In conventional shaken culture system, control of oxygen supply is performed by changing liquid volume in flasks and it necessarily introduces variation in the effectiveness of agitation and in the partial pressure of carbon dioxide. In jar or tank culture system, also, the changes in mechanical agitation and in the flow rate of air for control of aeration induce similar problems. It is impossible, therefore, to isolate the effects of oxygen on microbial metabolism from these accompanying ones. Hence, there is a basic requirement of making clear distinction among them, and in this paper the effects of agitation and carbon dioxide on product formation are presented in glutamic acid fermentation using the apparatus of controlling the level of dissolved oxygen throughout the fermentation.

To obtain fundamental knowledge required for attaining adequate aeration, the rate of oxygen demand in glutamic acid fermentation was discussed in connection with its fermentation rates. On the basis of specific rates, rates of change per unit mass of cells, glutamic acid fermentation was found to fall in the process pattern of Gaden’s type II, in which a constant rate of oxygen demand was sustained for a considerable time. On the basis of volumetric rates, rates of change per unit volume of broths, oxygen demand was recognized to be correlated with growth, sugar utilization and product formation, and it was pointed out particularly that the oxygen demand was closedly related with sugar utilization. In the particular cases where rapid utilization of sugar occurred, therefore, oxygen deficiency was liable to be evoked being unable to fill the growing oxygen demand. This finding might be useful for scale-up studies or process design.     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Der Respirationsfermentor – ein Labormeßsystem zur Charakterisierung aerober mikrobiologischer Reaktionen

By use of a laboratory fermenter with a gastight circle aeration system all interesting values connected with the respiration of microorganisms can be determined. The advantage of the respiration fermenter is the simple control of the oxygen partial pressure with high accuracy. The aeration is carried out by inert gas-oxygen-mixtures. This system was used to avoid errors in oxygen-concentration measurement and regulation by oxygen sensitive sensors in fermentation fluids. For each selected oxygen partial pressure in the course of fermentation the following date in connection with all desired analytical values can be determined by manual or automatical means. Total oxygen consumption, momentary oxygen consumption rate, kinetics of oxygen consumption rate, kinetics of respiratory quotient. Several opportunities of measurement are presented and examples for application in various microbial systems are given.     

Effect of partial pressure of solute oxygen on the intensity of respiration of cultures of antibiotic producers

It was shown possible to use feeding of hydrogen peroxide as a method for investigating the impact of dissolved oxygen concentrations on growth and development of microorganisms. The influence of pO2 on the respiration intensity was studied in penicillin- and erythromycin-producing cultures and it was found that dependence of the respiration intensity on pO2 had the form of a curve with saturation, at pO2 equal to zero the value of the culture respiration intensity being different from zero. A mathematical model accounting for the presence in the fermentation broth of microbial agglomerates with the average size depending on the agitation conditions is proposed for describing the relationships.     

Determination of external and internal mass transfer limitation in nitrifying microbial aggregates

In this article we present a study of the effects of external and internal mass transfer limitation of oxygen in a nitrifying system. The oxygen uptake rates (OUR) were measured on both a macro-scale with a respirometric reactor using off-gas analysis (Titrimetric and Off-Gas Analysis (TOGA) sensor) and on a micro-scale with microsensors. These two methods provide independent, accurate measurements of the reaction rates and concentration profiles around and in the granules. The TOGA sensor and microsensor measurements showed a significant external mass transfer effect at low dissolved oxygen (DO) concentrations in the bulk liquid while it was insignificant at higher DO concentrations. The oxygen distribution with anaerobic or anoxic conditions in the center clearly shows major mass transfer limitation in the aggregate interior. The large drop in DO concentration of 22-80% between the bulk liquid and aggregate surface demonstrates that the external mass transfer resistance is also highly important. The maximum OUR even for floccular biomass was only attained at much higher DO concentrations (approximately 8 mg/L) than typically used in such systems. For granules, the DO required for maximal activity was estimated to be >20 mg/L, clearly indicating the effects of the major external and internal mass transfer limitations on the overall biomass activity. Smaller aggregates had a larger volumetric OUR indicating that the granules may have a lower activity in the interior part of the aggregate.     

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     

Dissolved oxygen as principal parameter for conidia production of biocontrol fungi Trichoderma viride in non-Newtonian wastewater

Dissolved oxygen (DO) concentration was selected as a principal parameter for translating results of shake flask fermentation of Trichoderma viride (biocontrol fungi) to a fermenter scale. All fermentations were carried out in a 7.5 l automated fermenter with a working volume of 4 l. Fermentation performance parameters such as volumetric oxygen transfer coefficient (k _L a), oxygen uptake rate (OUR), rheology, conidia concentration, glucose consumption, soluble chemical oxygen demand, entomotoxicity and inhibition index were measured. The conidia concentration, entomotoxicity and inhibition index were either stable or improved at lower DO concentration (30%). Variation of OUR aided in assessing the oxygen supply capacity of the fermenter and biomass growth. Meanwhile, rheological profiles demonstrated the variability of wastewater during fermentation due to mycelial growth and conidiation. In order to estimate power consumption, the agitation and the aeration requirements were quantified in terms of area under the curves, agitation vs. time (rpm h), and aeration vs. time (lpm h). This simple and novel strategy of fermenter operation proved to be highly successful which can be adopted to other biocontrol fungi.     

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.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.     

Determination of CO₂ sensitivity of micro-organisms in shaken bioreactors. II. Novel online monitoring method

In the present study, a new online monitoring method for the determination of the CO? sensitivity of micro-organisms, based on the values of the respiration factors [OTR (oxygen transfer rate) and CTR (carbon dioxide transfer rate)], obtained by using the RAMOS (respiratory activity monitoring system) device considering a variety of aeration rates in the measuring flask, is investigated. Based on the data of the OTR, obtained by RAMOS under a variety of specific aeration rates, the proposed new method was developed as an online monitoring method for CO? sensitivity of micro-organisms in shaken bioreactors. A maximum accumulated CO? concentration of 12% was derived in applied methods, provided that the cultivation system is carried out under optimal conditions. Additionally, to predict these conditions, an unsteady-state gas transfer model in shaken bioreactors would be very advantageous. The data of OTR obtained using the RAMOS device were analysed and recalculated by a programme considering the calibration factor (Cf). The major advantage of the new method is the possibility to determine the metabolic activity, regardless of manual sampling.