Bubble bed reactor: A reactor design to minimize the damage of bubble aeration on animal cells

A new bubble aeration system was designed to minimize cell killing and cellular damage due to sparging. The residence time of the bubbles in the developed bubble bed reactor was prolonged dramatically by floating them in a countercurrent produced by an impeller. The performance of the new reactor bubble aeration system, implemented in a laboratory reactor, was tested in dynamic aeration experiments with an without cells. An efficiency up to 95% in oxygen transfer could be achieved, which enables a much lower gas flow rate compared with conventional bubble aeration reactors. The low gas flow rate is important to keep cell damage by bubbles as low as possible. A laser light sheet technique used to find the optimal flow pattern in the reactor. The specific power dissipation of the impeller is a good measure to predict cell damage in a turbulent flow. Typical values for the power dissipation measured in the bubble bed reactor were in the range of 0.002 to 0.013 W/kg, which is far below the critical limit for animal cells. The growth of a hybridoma cell line was studied in cell cultivation experiments. A protein-free medium without supplements such as serum or Pluronic F68 was used to exclude any effect of cell-protecting factors, No difference in the specific growth rate and the yield of the antibodies was observed in cell grown in the bubble free surface aeration in the spinner flask. In contrast to the spinner flask, however, the bubble bed reactor design could be scaled up. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of production of cloned glucoamylase under conditions of low aeration from recombinant yeast using a SUC2 promoter

The effect of aeration rate on the production of cloned glucoamylase in a recombinant yeast was investigated. This system consisted of Saccharomyces cerevisiae transformed with the 2 μ-based plasmid YEpSUCSTA which contains the SUC2 promoter, the STA signal sequence, and the STA structural gene. In contrast to typical yeast expression reports, high production of cloned glucoamylase was achieved at low aeration level (0·3 vvm). The recombinant yeast grown at 0·3 vvm aeration produced more glucoamylase (0·94 units/ml) than when grown at 0·0 vvm, 0·6 vvm, or 0·9 vvm (9·4, 1·4, and 3·1 times more, respectively). A high dissolved oxygen level early in the cultivation was important for cell growth and a low dissolved oxygen level during the production stage was important for glucoamylase production. In large scale processes for the production of recombinant proteins, the maintenance of aeration and dissolved oxygen at high levels is difficult and expensive. In this work, we have evaluated the coordination of oxygen level with growth and protein production and developed optimal conditions. Since a low aeration rate was optimal, our results demonstrate that the method described at the laboratory scale should be successfully applied at an industrial scale.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

A new easy method for determination of surface adhesion of phototrophic biofilms

Terrestrial cyanobacteria grow as phototrophic biofilms and offer a wide spectrum of interesting products. For cultivation of phototrophic biofilms different reactor concepts have been developed in the last years. One of the main influencing factors is the surface material and the adhesion strength of the chosen production strain. In this work a flow chamber was developed, in which, in combination with optical coherence tomography and computational fluid dynamics simulation, an easy analysis of adhesion forces between different biofilms and varied surface materials is possible. Hereby, differences between two cyanobacteria strains and two surface materials were shown. With longer cultivation time of biofilms adhesion increased in all experiments. Additionally, the content of extracellular polymeric substances was analyzed and its role in surface adhesion was evaluated. To test the comparability of obtained results from the flow chamber with other methods, analogous experiments were conducted with a rotational rheometer, which proved to be successful. Thus, with the presented flow chamber an easy to implement method for analysis of biofilm adhesion was developed, which can be used in future research for determination of suitable combinations of microorganisms with cultivation surfaces on lab scale in advance of larger processes.     

Pilot-scale reactor for aseptic solid-state cultivation

Summary A new 50 l pilot scale reactor was used to carry out solid-state cultivations in sterile conditions using a forced aeration system with a planetary agitation device. The medium containing wheat bran wetted with a glucose solution was kept sterile for 8 days at 32°C.     

Monoseptic cultivation of phototrophic microorganisms--development and scale-up of a photobioreactor system with thermal sterilization

The use of phototrophic microorganisms as sources of biological active substances in photoautotrophic and mixotrophic cultivation modes requires an adequate cultivation system with thermal sterilization. A corresponding photobioreactor system in the 10, 25 and 100 l scales was developed. This "Medusa"-photobioreactor system represents a concept based on the air-lift loop principle, whose working volume is irradiated by external light sources. The incident irradiation can be varied by a light control system. An effective CO(2)/O(2) gas exchange is enabled due to the efficient supply with process gas by several gas supply nozzles within the system and a large degassing surface. Using a model to describe the growth characteristics of the organisms, the volumetric irradiation coefficient I(DX) was defined as scale-up parameter. On this basis the scale-up from 1 l bubble columns to the 10 and 100 l scales was realized. The scale-up was performed successfully with Chlorella salina as model organism. A maximum biomass concentration of 7.89 g (dry weight) l(-1) at a maximum specific growth rate of 0.058 h(-1) and a yield of 35 mg l(-1) h(-1) was obtained in a batch cultivation in the 100 l scale under photoautotrophic conditions with an initial biomass concentration of approx. 0.03 g l(-1).     

Two-Level factorial screening of new plasmid/strain combinations for prodution of recombinant-DNA products

This article treats the basic problem of selection of experimental conditions for microbiological experiments for evaluation of newly isolated bacterial strains, mutants, or plasmid/strain combinations. For this purpose shake flask experiments in a 2(10-4)confounded factorial design at resolution IV with four blocks of 16 flasks were used. The design was used for testing of two new strain/plasmid combinations (E. coli MT 102/403-SD2 and W 3110/403-SD2) i.e., both strains with the same plasmid 403-SD2. Both strains were integrated in the design, so both strains were tested with nine factors (temperature, aeration, glucose, initial pH, pH regulation, reduced aeration, chloramphenicol, acetate, and glycerol). With both strains the interaction between initial pH and reduced aeration had a significant influence on the yield of the recombinant-DNA product nuclease. There was more than a factor of 10 between lowest and highest yield of product. In this interactive system the strains reacted differently. MT 102/403-SD2 had highest yields at high initial pH (8.4) and no reduction in aeration, whereas W 3110/403-SD2 had highest yields of nuclease at low initial pH (7.4) and reduced aeration (rubber stopper inserted after cultivation for 12 h). These data (and previous work) clearly demonstrate that it is impossible to suggest a simple set of experimental conditions for testing of new plasmid/strain combinations. It is clear that the exclusive application of a standardized growth technique e.g., LB-medium at 37 degrees C at an unspecified and uncontrolled aeration level, may lead to wrong conclusions on properties and potentials of now plasmid/strain combinations and may lead to rejection of useful strains or plasmids.     

Cultivation of Bacillus thuringiensis in an airlift reactor with wire mesh draft tubes

An aeration strategy was proposed for foam control in an airlift reactor with double wire mesh draft tubes. The airlift reactor was employed in the cultivation of Bacillus thuringiensis for thuringiensin production. The aeration strategy involved two situations. If the foam rose and touched the foam probe, the air flow rate was dropped to a low value for a certain period. However, if the DO value was already below 10% of the saturation when the air flow rate was dropped, the conventional foam control was employed. The production of thuringiensin based on the proposed strategy was up to 70% higher than that of using the conventional cultivation method with addition of antifoam agents for foam control.     

In vitro sponge fragment culture of Chondrosia reniformis (Nardo, 1847)

In vitro cultivation systems for sponges (Porifera) have to be developed to produce compounds of value in biotechnological processes. Organotypic culture attempts, which maintain or mimic the natural tissue structure, are promising ways towards a biotechnology of sponges. We used the Mediterranean species Chondrosia reniformis for sponge fragment in vitro cultivation. The species is common throughout the Mediterranean, easy to keep in aquariums and shows good recovery and regeneration after fragmentation. The regeneration process of the 50-80 mm(3) fragments lasted for several days and resulted in a rounded or ovoid body shape. The aquiferous system was reduced. Cells performed proliferation during the first weeks as we could demonstrate by 5-bromo-2'-deoxy-uridine (BrdU) incorporation. No proliferation could be demonstrated after a culture period of 3 months, but silicate uptake. Cellular density decreased with cultivation length, but collagen production increased. Fragments have been kept in culture up to 19 months. C. reniformis can be used as a model system to develop feeding strategies and evaluate the biotechnological potential of sponge fragment in vitro cultivation.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.     

Immobilized Growth of the Peridinin-Producing Marine Dinoflagellate Symbiodinium in a Simple Biofilm Photobioreactor

Products from phototrophic dinoflagellates such as toxins or pigments are potentially important for applications in the biomedical sciences, especially in drug development. However, the technical cultivation of these organisms is often problematic due to their sensitivity to hydrodynamic (shear) stress that is a characteristic of suspension-based closed photobioreactors (PBRs). It is thus often thought that most species of dinoflagellates are non-cultivable at a technical scale. Recent advances in the development of biofilm PBRs that rely on immobilization of microalgae may hold potential to circumvent this major technical problem in dinoflagellate cultivation. In the present study, the dinoflagellate Symbiodinium voratum was grown immobilized on a Twin-Layer PBR for isolation of the carotenoid peridinin, an anti-cancerogenic compound. Biomass productivities ranged from 1.0 to 11.0 g m^?2 day^?1 dry matter per vertical growth surface and a maximal biomass yield of 114.5 g m^?2, depending on light intensity, supplementary CO₂, and type of substrate (paper or polycarbonate membrane) used. Compared to a suspension culture, the performance of the Twin-Layer PBRs exhibited significantly higher growth rates and maximal biomass yield. In the Twin-Layer PBR a maximal peridinin productivity of 24 mg m^?2 day^?1 was determined at a light intensity of 74 μmol m^?2 s^?1, although the highest peridinin content per dry weight (1.7 % w/w) was attained at lower light intensities. The results demonstrate that a biofilm-based PBR that minimizes hydrodynamic shear forces is applicable to technical-scale cultivation of dinoflagellates and may foster biotechnological applications of these abundant marine protists.     

Microfluidic biolector—microfluidic bioprocess control in microtiter plates

In industrial‐scale biotechnological processes, the active control of the pH‐value combined with the controlled feeding of substrate solutions (fed‐batch) is the standard strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small‐scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale‐up and scale‐down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small‐scale batches are typically performed highly parallel and in high throughput, large‐scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber‐optic online‐monitoring device for microtiter plates (MTPs)—the BioLector technology—together with microfluidic control of cultivation processes in volumes below 1 mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed‐batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user‐friendly and can easily be transferred to a disposable single‐use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH‐controlled and fed‐batch conditions in shaken MTPs. Biotechnol. Bioeng. 2010;107: 497–505. © 2010 Wiley Periodicals, Inc.     

Effect of submerged culture conditions on exopolysaccharides production by Armillaria luteo-virens Sacc QH and kinetic modeling

This work aimed to develop the submerged cultivation conditions for improved exopolysaccharides (EPS) production by Armillaria luteo-virens Sacc. The effects of culture temperature, aeration rate, inoculum level, initial pH, and additives on EPS formation and mycelial growth are investigated. The aeration rate, initial pH, and inoculum level significantly affected EPS production under the submerged cultivation. The developed conditions were as follows: cultivation temperature 23 °C, initial pH 5.0, aeration rate 0.5 vvm, 0.5% Tween 80, inoculum level 5% (v/v), and shaking speed 120 r/min. Under the developed conditions, the highest EPS production was 13.01 g/L at 5 days culture time. EPS production was examined in a 5 L bioreactor, and an unstructured kinetic model for EPS formation was well developed. The verified investigations in the large-scale cultivation system showed that the developed models are able to predict the submerged cultivation process of EPS formation. Current results revealed that the submerged cultivation conditions can be utilized to control EPS production, and the unstructured models developed are suitable for explaining EPS production by A. luteo-virens Sacc QH in a large-scale cultivation bioreactor.     

Combination of algae and yeast fermentation for an integrated process to produce single cell oils

Economic and ecological reasons cause the industry to develop new innovative bio-based processes for the production of oil as renewable feedstock. Petroleum resources are expected to be depleted in the near future. Plant oils as sole substituent are highly criticized because of the competitive utilization of the agricultural area for food and energy feedstock production. Microbial lipids of oleaginous microorganisms are therefore a suitable alternative. To decrease production costs of microbial lipids and gain spatial independence from industrial sites of CO₂ emission, a combination of heterotrophic and phototrophic cultivation with integrated CO₂ recycling was investigated in this study. A feasibility study on a semi-pilot scale was conducted and showed that the cultivation of the oleaginous yeast Cryptococcus curvatus on a 1.2-L scale was sufficient to supply a culture of the oleaginous microalgae Phaeodactylum tricornutum in a 21-L bubble column reactor with CO₂ while single cell oils were produced in both processes due to a nutrient limitation.     

Characteristics of aerobic granular sludge in a sequencing batch reactor with variable aeration

Aerobic granules can be used for the treatment of industrial or municipal wastewater, but high aeration rate is required for the stable operation of the granular sludge system. Therefore, the aim of this research was to reduce aeration rate greatly to decrease the energy consumption for the technology of aerobic granules. Based on the characteristics of sequencing batch reactor with distinct feast and famine periods, aeration rate was reduced from 1.66 to 0.55 cm s⁻¹ in the famine period after granules were formed. It was found that the settleability of aerobic granules in reactor R1 with reduced aeration was the same as that of aerobic granules in reactor R2 with constant aeration rate of 1.66 cm s⁻¹. However, the outer morphology of aerobic granules gradually changed from round shape to long shape, and minor population showed certain shift after aeration rate was reduced in the famine period. Since good settleability is the most essential feature of aerobic granules, it can be said that reducing aeration rate in famine period did not influence the stable operation of aerobic granular sludge system. Furthermore, the experimental results indicated that aeration rate in feast period was much more important to the stable operation of aerobic granules than that in famine period.     

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter‐scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter‐scale. A newly designed one‐sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface‐to‐volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k_La) > 0.15 s^?1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter‐scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory‐scale stirred tank bioreactor with six‐bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter‐scale stirred tank bioreactor was reduced compared to the laboratory‐scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale‐up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter‐scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear‐thinning non‐Newtonian behavior. The newly developed one‐sided paddle impellers operated in unbaffled reactors on a 10 milliliter‐scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443–451. © 2010 Wiley Periodicals, Inc.     

Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.     

From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants

The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.     

Nitrogen transformations during biological aerobic treatment of pig slurry: effect of intermittent aeration on nitrous oxide emissions

A laboratory scale aeration treatment system was built to study the fate of nitrogen during aeration of pig slurry. For each run evaluated, the nitrogen mass balance was determined including measurement of the nitrous oxide gas emissions. Intermittent aeration led to a nitrogen removal of about 53% of the total nitrogen content of the raw slurry. About 18% of the total nitrogen content of the raw slurry was emitted as N2O during aeration with an aerobic to anoxic ratio equal to 0.625. In contrast, the extension of the anoxic period (aerobic to anoxic ratio = 0.375) allowed complete denitrification and avoided N2O emissions.