Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: use of citric acid as the carbon source

The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C_c = 9.0 kg m⁻³ citric acid as sole carbon source were investigated in 3.5 dm³ batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. At Q_o/V = 1 vvm air flow rate, the effect of agitation rate on DO concentration, pH, product, and by-product concentrations and SAP activity were investigated at N = 150, 500, and 750 min⁻¹; these are named as low-(LOT), medium-(MOT), and high oxygen transfer (HOT) conditions. LOT conditions favor biomass concentration; however, substrate consumption was highest at HOT conditions. MOT was optimum for maximum SAP activity which was 441 U cm⁻³ at t = 37 h. The total amino acid concentration was maximum in LOT and minimum in MOT conditions; lysine had the highest concentration under all oxygen transfer conditions. Among organic acids, acetic acid had the highest concentration and its concentration increased with oxygen transfer rate. The oxygen transfer coefficient increases with the agitation rate and the oxygen consumption rate increased almost linearly with the biomass concentration.     

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.     

Effect of oxygen transfer on glycerol biosynthesis by an osmophilic yeast Candida magnoliae I(2)B

The influence of oxygen on glycerol production by an osmophilic yeast, Candida magnoliae I(2)B, was studied in a bioreactor. Oxygen transfer rates (OTRs) and volumetric oxygen transfer coefficients (k(L)a) were determined at different aeration and agitation rates. Cell growth as well as glycerol production was strongly affected by oxygen supply. Improvement in OTRs resulted in increased cell growth and glycerol yield. However, at high OTRs, there was a reduction in glucose uptake rate, indicating Pasteur Effect, and glycerol accumulation was also reduced at k(L)a of 253 h(-1). The availability of oxygen per unit of cell mass was found to be the most important factor that controlled cell growth, glucose uptake, and glycerol yield. The overall productivity and yield of glycerol could be related with k(L)a. The biosynthesis of glycerol was found to both growth- and non-growth-associated, although glycerol was mainly produced in post-exponential phase.     

以摇瓶所得摄氧率为基准进行发酵放大

通过设计特殊摇瓶,用亚硫酸盐法测出摇瓶口纱布层氧通透率的基础上,在实际发酵情况下通过测定瓶内气、液相氧的变化得出其发酵过程中的摄氧率(OUR)及氧传递系数(K_La)。以特制摇瓶取得的菌体CUR为基准进行发酵过程和发酵罐的放大。通过质谱仪在线检测及采样分析,研究了3种不同供气流量及搅拌转速下的放大结果。摇瓶与发酵罐在菌体OUR、菌体产量方面吻合很好,而在整个放大过程中,发现摇瓶与发酵罐内的氧传递系数(K_La)、溶解氧(C_L)差异较大。     

Determination of specific oxygen uptake rate of Photorhabdus luminescens during submerged culture in lab scale bioreactor

Photorhabdus luminescens, a bacterial symbiont of entomoparasitic nematodes, was cultured in a 10 L bioreactor. Cellular density and bioluminescence were recorded and volumetric oxygen transfer coefficient (k_La) and specific oxygen transfer rates were determined during the batch process. Exponential phase of the bacterium lasted for 20 h, showing a maximum specific growth rate of 0.339 h^?1 in a defined medium. Bioluminescence peaked within 21h, and was maintained until the end of the batch process (48 h). The specific oxygen uptake rate (SOUR) was high during both lag and early exponential phase, and eventually reached a stable value of 0.33 mmol g^?1 h^?1 during stationary phase. Maintenance of 200 rpm agitation and 1.4 volume of air per volume of medium per minute (vvm) aeration, gave rise to a k_La value of 39.5 h^?1. This k_La value was sufficient to meet the oxygen demand of 14.4 g L^?1 (DCW) biomass. This research is particularly relevant since there are no reports available on SOURs of symbiotic bacteria or their nematode partners. The insight gained through this study will be useful during the development of a submerged monoxenic culture of Heterorhabditis bacteriophora and its symbiotic bacterium P. luminescens in bioreactors.     

Analyzing the suitability of a baffled orbitally shaken bioreactor for cells cultivation using the computational fluid dynamics approach

Orbitally shaken bioreactors (OSRs) is one of important bioreactors for mammalian cells cultivation in suspension, especially for the screening of valuable microorganisms and in basic bioprocess development experiments. However, the suitability of OSRs for cells culture in large scale is still under development. In this article, a new kind of OSRs with baffle structure was proposed and a three-dimensional CFD model was established to analyze the influence of baffle structure on the flow field. Lower installation height of baffles was found suitable for improving the mixing efficiency. Compared to the unbaffled OSR, the baffled OSR could enhance the level of oxygen transfer largely but the oxygen transfer rate was independent on the baffle installation height. Moreover, as the baffle installation height increased, the energy transferred for liquid motion was decreased. Finally, the shear stress of the baffled OSRs proposed was gentle for mammalian cells growth. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2746, 2019     

Characterization of gas-liquid mass transfer phenomena in microtiter plates

Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L). An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells.     

Effects of air pressure oscillation amplitude on oxygen transfer rate and biomass productivity in a solid-state fermenter

The modified sulfite oxidation method was adapted for estimation of the overall oxygen transfer rate in a pressure oscillating, solid-state fermenter. At 4.5 atm and 30 °C, the oxygen transfer rate reached 717 mmol kg^–1 initial dry matter h^–1 in this system against 37 mmol kg^–1 initial dry matter h^–1 in a static tray fermenter. At 30 °C and 3 atm, Azotobacter vinelandii grew on wheat straw and reached 4.7×10¹⁰ c.f.u. g^–1 substrate dry matter after 36 h, while only 8.2×10⁹ c.f.u. g^–1 substrate dry matter was obtained in a static tray system.     

A dense cell retention culture system using stirred ceramic membrane reactor

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the k(L)a to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed-batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. (c) 1994 John Wiley & Sons, Inc.     

Effects of the oxygen transfer rate on ferrous iron oxidation by Thiobacillus ferrooxidans

Ferrous iron oxidation by Thiobacillus ferrooxidans was studied in shake flasks and a bubble column under different aeration conditions. The maximum biooxidation rate constant was affected by oxygen transfer only at low aeration intensities. At oxygen transfer rates higher than 0.03 mmol O₂ l⁻¹ min⁻¹, the maximum biooxidation rate constant was about 0.050 h⁻¹ in both shake flasks of different size and the bubble column. The oxygen transfer rate could be used as a basis for scaling up bioreactors for ferrous iron biooxidation by T. ferrooxidans.     

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.     

Effective diffusivity of oxygen in microbial pellets

In a typical submerged aerobic fermentation with microbial pellets, the effective diffusivity of oxygen in the pellets is probably the most important, yet most difficult transport property to characterize experimentally. Its values directly indicate the efficiency or deficiency of oxygen to individual cells, and thus the biological activity of the microorganisms. In the past, it was not possible to assess reliably the effective diffusivity of oxygen in pellets due to several reasons. Firstly, most oxygen electrodes available were coarse, and hence not suitable for in situ measurements. Secondly, there was a lack of methods rigorous enough to characterize the structure of the microbial pellets. A state-of-the-art review of the literature relating to the feature subject is presented. Emphasis is laid upon development and evolution of the means for quantitative characterization of the effective diffusivity of oxygen in microbial pellets.     

Over‐pressurized bioreactors: Application to microbial cell cultures

In industrial biotechnology, microbial cultures are exposed to different local pressures inside bioreactors. Depending on the microbial species and strains, the increased pressure may have detrimental or beneficial effects on cellular growth and product formation. In this review, the effects of increased air pressure on various microbial cultures growing in bioreactors under moderate total pressure conditions (maximum, 15 bar) will be discussed. Recent data illustrating the diversity of increased air pressure effects at different levels in microbial cells cultivation will be presented, with particular attention to the effects of oxygen and carbon dioxide partial pressures on cellular growth and product formation, and the concomitant effect of oxygen pressure on antioxidant cellular defense mechanisms. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:767–775, 2014     

Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth

Oxygen limitation is one of the most frequent problems associated with the application of shaking bioreactors. The gas-liquid oxygen transfer properties of shaken 48-well microtiter plates (MTPs) were analyzed at different filling volumes, shaking diameters, and shaking frequencies. On the one hand, an optical method based on sulfite oxidation was used as a chemical model system to determine the maximum oxygen transfer capacity (OTR(max)). On the other hand, the Respiration Activity Monitoring System (RAMOS) was applied for online measurement of the oxygen transfer rate (OTR) during growth of the methylotropic yeast Hansenula polymorpha. A proportionality constant between the OTR(max) of the biological system and the OTR(max) of the chemical system were indicated from these data, offering the possibility to transform the whole set of chemical data to biologically relevant conditions. The results exposed "out of phase" shaking conditions at a shaking diameter of 1 mm, which were confirmed by theoretical consideration with the phase number (Ph). At larger shaking diameters (2-50 mm) the oxygen transfer rate in MTPs shaken at high frequencies reached values of up to 0.28 mol/L/h, corresponding to a volumetric mass transfer coefficient (k(L)a) of 1,600 1/h. The specific mass transfer area (a) increases exponentially with the shaking frequency up to values of 2,400 1/m. On the contrary, the mass transfer coefficient (k(L)) is constant at a level of about 0.15 m/h over a wide range of shaking frequencies and shaking diameters. However, at high shaking frequencies, when the complete liquid volume forms a thin film on the cylindric wall of the well, the mass transfer coefficient (k(L)) increases linearly to values of up to 0.76 m/h. Essentially, the present investigation demonstrates that the 48-well plate outperforms the 96-well MTP and shake flasks at widely used operating conditions with respect to oxygen supply. The 48-well plates emerge, therefore, as an excellent alternative for microbial cultivation and expression studies combining the advantages of both the high-throughput 96-well MTP and the classical shaken Erlenmeyer flask.     

The impact of influent nutrient ratios and biochemical reactions on oxygen transfer in an EBPR process--a theoretical explanation

In this investigation, a laboratory-scale enhanced biological phosphorus removal (EBPR) process was operated under controlled conditions to study the impact of varying the influent ratio of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN) and total phosphorus (TP), and the consequential biochemical reactions on oxygen transfer parameters. The data showed that the experiment with high influent phosphorus relative to nitrogen (COD/TP = 51 and TKN/TP = 3.1) achieved higher alpha and oxygen transfer efficiency (OTE(f)). On the other hand, the experiment with high influent nitrogen relative to phosphorus (TKN/TP = 14.7 and COD/TP = 129) resulted in approximately 50% reduction in alpha and OTE(f) under similar organic loading. This suggested that the intracellular carbon storage and the enhanced biological P removal phenomenon associated with the phosphorus-accumulating organisms (PAOs) had a positive influence on OTE(f) in the high phosphorus experiment compared to an active population of nitrifying and denitrifying organisms in the high nitrogen experiment. The intracellular carbon storage by the glycogen-accumulating organisms also appeared to have had a positive effect on oxygen transfer efficiency, although to a lesser extent in comparison to the PAOs. It was also found that oxygen uptake rate (OUR) was not a good indicator of the measured alpha and OTE(f), because it was a combined effect of several biochemical reactions, each having a varying degree of influence. It is difficult to underestimate the crucial role of flocs in mass transfer of oxygen, because microorganisms associated with flocs carry out the biochemical reactions. It seems that the combination of influent characteristics and biochemical reactions in each experiment produced a unique biomass quality (determined by the biomass N to P ratio), ultimately affecting the mass transfer of oxygen. A theoretical explanation for the observed oxygen transfer efficiency under the process conditions is also proposed in this article.     

Oxygen transfer characteristics of miniaturized bioreactor systems

Since their introduction in 2001 miniaturized bioreactor systems have made great advances in function and performance. In this article the dissolved oxygen (DO) transfer performance of submilliliter microbioreactors, and 1–10 mL minibioreactors was examined. Microbioreactors have reached k_La values of 460 h^?1, and are offering instrumentation and some functionality comparable to production systems, but at high throughput screening volumes. Minibioreactors, aside from one 1,440 h^?1 k_La system, have not offered as high rates of DO transfer, but have demonstrated superior integration with automated fluid handling systems. Microbioreactors have been typically limited to studies with E. coli, while minibioreactors have offered greater versatility in this regard. Further, mathematical relationships confirming the applicability of k_La measurements across all scales have been derived, and alternatives to fluorescence lifetime DO sensors have been evaluated. Finally, the influence on reactor performance of oxygen uptake rate (OUR), and the possibility of its real‐time measurement have been explored. Biotechnol. Bioeng. 2013; 110: 1005–1019. © 2012 Wiley Periodicals, Inc.     

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high‐cell‐density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth‐limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium‐chain‐length polyhydroxyalkanoate (mcl‐PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ～30 and ～90 mg L^?1, respectively. Remarkably, a little increase of mcl‐PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high‐cell‐density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl‐PHA content occurred up to a DCT of ～540 mg L^?1. However, specific growth rate exhibited a significant decrease, indicating that successful high‐cell‐density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed‐batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl‐PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl‐PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L^?1 h^?1 (at 3.2 bar). Biotechnol. Bioeng. 2012; 109:451–461. © 2011 Wiley Periodicals, Inc.