Biomedical subjects

H Märkl

Publications and source records attributed to H Märkl.

18 recordsLinked to original sources

PubMed2003

Improvement of a mammalian cell culture process by adaptive, model-based dialysis fed-batch cultivation and suppression of apoptosis.

Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products. In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented. On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis. Maximum cell density could be increased by a factor of approximately 5-7 by dialysis fed-batch cultivation. On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3. These factors resulted in more product formation (by a factor of approximately 2). Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.

Adaptation, Physiological↗

PubMed2003

Biodegradation of aliphatic and aromatic hydrocarbons at high temperatures.

In this paper, the high temperature (65-75 degrees C) biodegradation of aliphatic and aromatic hydrocarbons is investigated and kinetic parameters are derived. The shift of the physico-chemical system properties with rising temperature will be discussed in detail. For example, the solubility of naphthalene is increased by a factor of about ten if the temperature is increased from 20 to 75 degrees C. This effect is essential to increase the bioavailability of sparingly soluble hydrocarbons. It is also demonstrated in experiments that very high oxygen transfer rates can be obtained at high temperatures in the presence of hydrocarbons. It is shown that efficient phenol biodegradation is essential for high temperature hydrocarbon degradation because some microorganisms tend to transform phenols into polyphenols which are very inhibitory for microbial growth. A defined mixed culture adapted to phenol converted more than 90% of a mixture of phenol, hexadecane and pyreno and a very high maximal growth rate of 0.19 h(-1) was determined. A yield coefficient Y(X/S) of about 0.8 g (biomass)/g (hydrocarbons) was calculated in this experiment. In a separate experiment the influence of the hydrocarbon droplet size on the biodegradation is investigated at 70 degrees C using a newly isolated Thermus sp. In this case, the growth on a hexadecane/pyrene mixture was described by a model based on the Monod equation and the corresponding kinetic parameters are derived. A mixed culture was used for the bioremediation of soil in a slurry reactor. The initial contamination of 11 g/kg was lowered to about 2 g in a reactor inoculated by an immobilized culture of extreme thermophilic microorganisms, while 9 g/kg remained in a sterile control.

Biodegradation, Environmental↗

PubMed2003

Airlift-reactor design and test for aerobic environmental bioprocesses with extremely high solid contents at high temperatures.

Bioprocesses at high temperatures gained considerably in importance within the last years and several new applications for aerobic, extreme thermophilic environmental bioprocesses are emerging. However, this development is not yet matched by adequate bioreactor designs, especially if it comes to the treatment of solids. In this communication we propose the use of airlift reactors to bridge this gap. The design of an internal draught tube bioreactor (Area(Riser)/Area(Downcomer) . 1; Height/Diameter . 8) is described in detail. The influence of the temperature on gas hold-up, liquid velocity and mixing characteristics was investigated. It was shown that this reactor could hold up to 1 t quartz sand per m3 in suspension at moderate aeration rates. Despite the decreasing oxygen solubility, the oxygen transfer rate increased with rising temperature due to the improved mass transfer parameters. With rising solid content, the oxygen transfer rate increased and reached a maximum at a solid content of about 140 kg m(-3) before it decreased again. However, it is only slightly reduced at the highest solid contents. The results demonstrate that aerobic bioprocesses at high temperatures are not only feasible, but can be very efficient if carried out in proper bioreactors.

Bacteria, Aerobic↗

PubMed2002

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.

Culture Media↗

PubMed2002

In-situ-fluorescence-probes: a useful tool for non-invasive bioprocess monitoring.

Optical sensors appear to be very promising for different applications in modern biotechnology. They offer the possibility to interface all the well known optical analysis techniques to bioprocesses via fiber optical cables. Thus, high sophisticated and sensitive optical analysis techniques can be coupled to a bioprocess via these light signal transporting fibers. A wide variety of sensor types for application in biotechnology has been described. Normally these sensors are non-invasive and the response times are nearly instantaneous. In particular, the use of glass fiber technology makes these sensors small, robust and reduces their costs.

Biomass↗

PubMed2001

Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2.

Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications.

Bacillus↗

PubMed2000

Modeling of olive oil degradation and oleic acid inhibition during chemostat and batch cultivation of Bacillus thermoleovorans IHI-91.

Olive oil degradation by the thermophilic lipolytic strain Bacillus thermoleovorans IHI-91 in chemostat and batch culture was modeled to obtain a general understanding of the underlying principles and limitations of the process and to quantify its stoichiometry. Chemostat experiments with olive oil as the sole carbon source were successfully described using the Monod chemostat model extended by terms for maintenance requirements and wall growth. Maintenance requirements and biomass yield coefficients were in the range reported for mesophiles. For a chemostat experiment at D = 0.3 h(-1) the model was validated up to an olive oil feed concentration of about 3.0 g L(-1) above which an inhibitory effect occurred. Further analysis showed that the liberated oleic acid is the main cause for this inhibition. Using steady-state oleic acid concentrations measured in chemostat experiments with olive oil as substrate it was possible to derive a kinetic expression for oleic acid utilization, showing that a concentration of 430 mg L(-1) leads to a complete growth inhibition. Oleic acid accumulation observed during batch fermentations can be predicted using a model involving growth-associated lipase production and olive oil hydrolysis. Simulations confirmed that this accumulation is the cause for the sudden growth cessation occurring in batch fermentations with higher olive oil start concentrations. Further, an oscillatory behavior, as observed in some chemostat experiments, can also be predicted using the latter model. This work clearly demonstrates that thermophilic lipid degradation by Bacillus thermoleovorans IHI-91 is limited by long-chain fatty acid beta-oxidation rather than oil hydrolysis.

Bacillus↗

PubMed2000

Isolation and characterization of lipid-degrading Bacillus thermoleovorans IHI-91 from an icelandic hot spring.

An efficient lipid-degrading thermophilic aerobic bacterium was isolated from an icelandic hot spring and classified as Bacillus thermoleovorans IHI-91. The aerobic bacterium grows optimally at 65 degrees C and pH 6.0 and secretes a high level of lipase (300 Ul(-1)). The newly isolated strain utilizes several lipids such as palmitic acid, stearic acid, lanolin, olive oil, sunflower seed oil, soya oil, and fish oil as sole carbon and energy source without an additional supply of growth factors. The degradation of about 93% of triolein, which is present in olive oil, was observed after only 7h of fermentation at a maximal growth rate of 1.0 h(-1). During growth at optimal conditions on yeast extract, the doubling time was only 15 min. Based on 16S rDNA studies, DNA-DNA hybridization and morphological and physiological properties, the isolate IHI-91 was identified as Bacillus thermoleovorans IHI-91 sp. nov. Because of its production of high concentrations of thermoactive lipases and esterases and the capability of degrading a wide range of lipids at high temperatures, the isolated strain is an ideal candidate for application in various biotechnological processes such as wastewater treatment.

Bacillus↗

PubMed1998

Dialysis cultures.

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a "nutrient-split" feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective.

Animals↗

PubMed1997

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil.

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 degrees C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l-1 h-1 at a condition rate of 0.4 h-1. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant Ks of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, mu max, of 1.0 h-1. Oxygen uptake rates of up to 2.9 g l-1 h-1 were calculated and the yield coefficient, Y X/O, was determined to be 0.29 g dry cell weight/g O2. From an overall material balance the yield coefficient, Y X/S, was estimated to be 0.60 g dry cell weight/g olive oil.

Bacillus↗

PubMed1997

Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor.

High-cell-density cultivations of Escherichia coli K12 in a dialysis reactor with controlled levels of dissolved oxygen were carried out with different carbon sources: glucose and glycerol. Extremely high cell concentrations of 190 g/l and 180 g/l dry cell weight were obtained in glucose medium and in glycerol medium respectively. Different behaviour was observed in the formation of acetic acid in these cultivations. In glucose medium, acetic acid was formed during the earlier phase of cultivation. However, in glycerol medium, acetic acid formation started later and was particularly rapid at the end of the cultivation. In order to estimate the influence of acetic acid during these high-cell-density cultivations, the inhibitory effect of acetic acid on cell growth was investigated under different culture conditions. It was found that the inhibition of cell growth by acetic acid in the fermentor was much less than that in a shaker culture. On the basis of the results obtained in these investigations of the inhibitory effect of acetic acid, and the mathematical predictions of cell growth in a dialysis reactor, the influence of acetic acid on high-cell-density cultivation is discussed.

Acetic Acid↗

PubMed1996

Killing of microorganisms by pulsed electric fields.

Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more than four orders of magnitude (> 99.99%). On the other hand, endo- and ascospores were not inactivated or killed to any great extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time (the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric field strength (Ec) and a specific critical treatment time (tc) were determined. Above these critical values, the fractions of surviving cells were reduced drastically. The "limits" Ec and tc depend on the cell characteristics as well as on the type of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied. Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of microbial cells.

Bacillus cereus↗

PubMed1995

Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line.

A bioreactor system for the continuous cultivation of animal cells with a high potential for scale-up is presented. This reactor system consists of radial-flow fixed-bed units coupled with a dialysis module The dialysis membrane enables the supply of low-molecular-weight nutrients and removal of toxic metabolites, while high-molecular-weight nutrients and products (e.g., monoclonal antibodies) are retained and accumulated. This concept was investigated on the laboratory scale in a bioreactor with an integrated dialysis membrane. The efficiency of the reactor system and the reproducibility of the cell activity (hybridoma cells) under certain process conditions could be demonstrated in fermentations up to 77 days. Based on model calculations, an optimized fermentation strategy was formulated and experimentally confirmed. Compared to chemostat cultures with suspended cells, a ten-times higher mAb concentration (383 mg1(-1)) could be obtained. The highest volumetric specific mAb production rate determined was 6.1 mg mAb (1 fixed bed)-1h-1.

Animals↗

PubMed1994

Estimation of specific glucose uptake rates in cultures of hybridoma cells.

The specific glucose uptake rate of a hybridoma cell line was determined in batch and continuous suspension cultures. For high density cultivation a membrane dialysis reactor was used. The aim was to obtain data, which can be used for process design and control. A decrease of the specific glucose uptake rate was observed during the course of batch cultures, even when the specific growth rate remained constant. These results are valid for cell densities between 2 x 10(5) and 2 x 10(6) ml-1. At cell densities between 2 x 10(6) and 1.5 x 10(7) ml-1 during continuous cultivation the specific glucose uptake rate remained constant. A relationship between the specific glucose uptake rate and the cell number was found and formulated in a mathematical equation. Literature data for different hybridoma cell lines fit in this plot very well. Applying this relationship, a correct estimation of the course of the glucose concentration in batch and continuous cultivation is possible.

Animals↗

PubMed1994

High density cultivation of hybridoma in charged porous carriers.

A porous carrier whose surface is charged with polyethyleneimine (PEI) was evaluated for high-density cultivation of immobilized hybridoma cells. The carrier was used either in suspension or in fixed bed. For suspended carriers, the effect of carrier pore size, ion-exchange capacity (IEC), mixing intensity and inoculum concentration on the attachment and growth of HBs MAb-producing hybridoma cells in carrier CELLSNOW EX were studied. The carrier with 100 microns pore size was more efficient than that with 500 microns in supporting cell attachment. In addition, the amount of antibody which was detected by PHA (passive hemagglutination assay) was 4-times higher in 100 microns compared with 500 microns pore size. At low initial cell concentration, 2.8 x 10(5) ml-1, the attached cells did not grow in the first 7 d as indicated by a negligible glucose consumption. However, cell proliferation was observed at high inoculum, 9 x 10(5) ml-1. Hybridoma cells were expected to attach faster to EX with high IEC than with low IEC but results showed no significant difference. A different hybridoma cell line producing monoclonal antibody (MAb) against penicillin-G-amidase was successfully cultivated in an axial-flow fixed bed containing CELLSNOW carriers. The specific glucose uptake rate as an indicator for cell activity was 24-times higher compared with conventional chemostat cultures. The CELLSNOW carriers offers many advantages over other carriers.

Animals↗

PubMed1994

Effect of NH3 on the cell growth of a hybridoma cell line.

Ammonia often has been reported to inhibit cell growth. The aqueous ammonia equilibrium between the un-ionized from (NH3) and the ammonium ion (NH4+) depends on the pH of the solution. Extensive studies in batch and continuous cultivation by varying pH and total ammonia concentration were carried out to investigate whether a kinetic model describing growth inhibition by ammonia has to be based on the total ammonia concentration, or the concentration of NH3. A significant relationship between the specific growth rate and death rate, respectively, and the NH3 concentration, but not the total ammonia concentration, was detected. An adaptation of the cells to high ammonia levels was not observed. Based on these results a new kinetic model for ammonia mediated growth inhibition is suggested. For high density cultivation it is recommended to control the pH at the lower limit of the growth optimum to keep the NH3 level low.

Ammonia↗

PubMed1993

Cultivation of Escherichia coli to high cell densities in a dialysis reactor.

High cell density cultivation of Escherichia coli on a glycerol-based mineral medium was studied. The cultivation was done in a dialysis reactor composed of two chambers. The inner chamber is formed and separated from an outer chamber by a membrane. Fresh medium was continuously exchanged with medium in the outer chamber so that both glycerol and other components of the medium were supplied to the inner chamber through the membrane. Inhibitory substances diffused from the inner to the outer chamber and were subsequently removed with effluent from the outer chamber. Initially, mathematical models were used to describe the process. The optimal cultivation parameters, such as the initial glycerol concentrations in the two chambers, the desired transport rate across the membrane, glycerol concentration in the feed/dialysing medium, and the time to start the medium exchange, were determined from preliminary experiments and calculations. The actual cultivation results agreed very well with the model predictions. A very high cell concentration of 174 g dry weight/l was obtained. This cell concentration is within the range of the maximum theoretical concentration of E. coli in culture broth (160-200 g/l).

Cell Division↗

PubMed1992

The membrane dialysis bioreactor with integrated radial-flow fixed bed--a new approach for continuous cultivation of animal cells.

A hybridoma cell was cultivated continuously in a membrane dialysis bioreactor with an integrated radial-flow fixed bed consisting of porous Siran carriers over a period of 6 weeks. Antibodies accumulated to an average of 100 mg l-1, approx. 10 times more than in fixed bed cultures without dialysis membrane. Serum costs could be reduced about 85% due to an appropriate feeding strategy. Siran carriers with 3-5 mm diameter showed an advantage compared to those with 1-2 mm diameter. For the 3-5 mm carrier the specific glucose uptake rate and the MAb production rate were constant, if the velocity was between 0.09 mm s-1 and 0.75 mm s-1. At higher velocities cells are washed out of the bed. Furthermore antibody consistency and cell stability were verified in long-term cultivations over a period of 96 days. From an estimation of the antibody concentration reachable with the reactor concept under optimal conditions a concentration 45 times higher compared to axial-flow fixed bed reactors and 11 times higher compared to stirred tank reactors can be expected.

Animals↗