Characterisation of a new thermoalkaliphilic bacterium for the production of high-quality hemp fibres, Geobacillus thermoglucosidasius strain PB94A.

Novel thermophilic and alkaliphilic bacteria for the processing of bast fibres were isolated using hemp pectin as substrate. The strain PB94A, which showed the highest growth rate (micro = 0.5/h) was identified as Geobacillus thermoglucosidasius (DSM 21625). The strain grew optimally at 60 degrees C and pH 8.5. During growth on citrus pectin, the strain produced pectinolytic lyases, which were excreted into the medium. In contrast to the commercially available pectinase Bioprep 3000 L, the enzymes from G. thermoglucosidasius PB94A converted pectin isolated from hemp fibres. In addition to hemp pectin, the culture supernatant also degraded citrus, sugar beet and apple pectin and polygalacturonic acid. When hemp fibres were incubated with the cell-free fermentation broth of G. thermoglucosidasius PB94A, the fineness of the fibres increased. The strain did not produce any cellulases, which is important in order to avoid damaging the fibres during incubation. Therefore, these bacteria or their enzymes can be used to produce fine high-quality hemp fibres.

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.

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.

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.

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.

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.

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.

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.

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.

Phytotoxicities of Selected Chemicals and Industrial Effluents to Nitellopsis obtuse Cells, Assessed by Using a Rapid Electrophysiological Charophyte Test.

The acute phytotoxicities of seven heavy metals (Cd2+, Cu2+, Hg2+, Ni2+, Zn2+, Cr6+ and Co2+), three phenolic compounds (phenol, 3,5-dichlorophenol and pentachlorophenol) and nine industrial effluents were appraised by using a rapid electrophysiological test with cells of the charophyte, Nitellopsis obtusa. The EC50 values (concentrations causing a 50% decrease in resting potential) obtained for reference chemicals were compared with those of five microbiotests (Polytox®, Microtox®, Selenastrum capricornutum growth inhibition, Daphnia magna immobilisation and Rotoxkit F™) taken from the scientific literature. The 45-minute charophyte test, the freshwater Algaltoxkit F™, Daphtoxkit F™ and Rotoxkit F™ were conducted simultaneously to assess the toxicities of effluents. The Toxkit microbiotests were typically two orders of magnitude more sensitive than the electrophysiological charophyte test to pure chemicals. The electrophysiological charophyte test was generally more sensitive than the Toxkit microbiotests to complex effluents. The rapid electrophysiological test, employing the 45-minute membrane depolarisation of N. obtusa cells as an endpoint, demonstrated similar sensitivity to heavy metals and phenolic compounds as the 20-minute bacterial Polytox® test, but less sensitivity than the 15-minute Microtox® test. Therefore, this rapid macroalgal test appears to be valuable as a sublethal toxicity screening tool for effluents.

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.

[Human cloning. The biological fundamentals and an ethical-legal assessment]. / La clonación humana. Fundamentos biológicos y valoración ético-jurídica.

Following a description of the cloning process and how this might be used in humans, the authors examine the possibility of human cloning in the light of recognised ethical principles. They also address the question of whether current national and international laws are sufficient to prevent such practices.

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.

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies.

UNLABELLED: An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new "nutrient-split" feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l(-1) was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system. ABBREVIATIONS: c(Glc) - glucose concentration, mmol l(-1); c(Gln) - glutamine concentration, mmol l(-1); c(Amm) - ammonia concentration, mmol l(-1); c(Lac) - lactate concentration, mmol l(-1); c(MAb) - MAb concentration, mg l(-1); D - dilution rate, d(-1); D(i) - dilution rate in the inner chamber of the membrane dialysis reactor, d(-1); D(0) - dilution rate in the outer chamber of the membrane dialysis reactor, d(-1); q*(FB,Glc) - volume specific glucose uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1); q*(FB,Gln) - volume specific glutamine uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1).

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.

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.

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.