Herbert Märkl

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, endoand 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.

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

Abstract A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 °C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l−1 h−1 at a dilution 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, μmax, of 1.0 h−1. Oxygen uptake rates of up to 2.9 g l−1h−1 were calculated and the yield coefficient, YX/O, was determined to be 0.29 g dry cell weight/g O2. From an overall material balance the yield coefficient, YX/S, was estimated to be 0.60 g dry cell weight/g olive oil.

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

Abstract 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.

Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 6070 °C by Thermus and Bacillus spp

Although polycyclic aromatic hydrocarbons (PAH) and alkanesare biodegradable at ambient temperature, in some cases low bioavailabilities are thereason for slow biodegradation. Considerably higher mass transfer rates and PAH solubilities and hence bioavailabilities can be obtained at higher temperatures. Mixed and pure cultures of aerobic, extreme thermophilic microorganisms (Bacillus spp., Thermus sp.) were used to degrade PAH compounds and PAH/alkane mixtures at 65 °C. The microorganismsused grew on hydrocarbons as sole carbon and energy source. Optimal growthtemperatures were in the range of 60–70 °C at pH values of 6–7. The conversion of PAH with 3–5 rings (acenaphthene, fluoranthene, pyrene, benzo[e]pyrene) was demonstrated. Efficient PAH biodegradation required a second, degradable liquid phase. Thermus brockii Hamburg metabolized up to 40 mg (l h)-1 pyrene and 1000 mg(1 h)-1 hexadecane at 70 °C. Specific growth rates of 0.43 h-1 were measured for this strain with hexadecane/pyrene mixtures as the sole carbon and energy source in a 2-liter stirred bioreactor. About 0.7 g cell dry weight were formed from 1 g hydrocarbon. The experiments demonstrate the feasibility and efficiency of extreme thermophilic PAH and alkane biodegradation.

Lipase production of Staphylococcus carnosus in a dialysis fermentor

SummaryIn a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.

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

Abstract 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°C and pH 6.0 and secretes a high level of lipase (300 U l−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 7 h 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.

Thermophiles and fermentation technology

Abstract Thermophilic microorganisms have been of great scientific interest for several decades, principally in regard to their biotechnological potential and also of the thermostable enzymes they produce. Optimal cultivation techniques for these organisms are required, therefore, not only for basic study but also for evaluation of their thermostable microbial products. Operating a fermentor at elevated temperatures may be advantageous in terms of increased solubility of substrates, improved mass transfer due to decreased viscosity, and increased diffusion rates. However, the cultivation of thermophiles also has many associated problems. A high cultivation temperature can give unexpected problems affecting the choice of reactor design and construction materials, and with the heating and cooling of the fermentor. Other problems may be caused by the low solubility of gases and the instability of substrates and other reagents used. Furthermore, high productivity requires high cell densities to be achieved and in many cases thermophiles are characterised by low growth rates, low growth yields and susceptibility to substrate and product inhibition at low concentrations. Different ways to circumvent some of these problems, such as using gas-lift fermentors, dialysis fermentors or cultivation with cell recycling are discussed.

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/1 was obtained. This cell concentration is within the range of the maximum theoretical concentration of E. coli in culture broth (160–200 g/l).

On-line monitoring of organic substances with high-pressure liquid chromatography (HPLC) during the anaerobic fermentation of waste-water

The simultaneous determination of betaine (trimethyl-glycine), N,N-dimethylglycine (N,N-DMG), acetic, and propionic acid in complex organic matrices for process monitoring is described. In some cases also ammonia and chloride were detected. The determinations were carried out by means of isocratic cation-exchange chromatography. For the detection of betain and N,N-DMG an ultraviolet detector was used. All other substances were detected by conductivity. Linear calibration curves were obtained for betaine (0.01–7.71 g/l), both organic acids (0.06–60 mM), chloride (0.01–4.31 g/l), and ammonia (0.05–1.02 g/l) over the concentration range given in parentheses. The results obtained by HPLC were checked by gas-chromatographic (GC) measurements for the organic acids and by photometric tests for betaine and ammonia. The values for the organic acids obtained by HPLC and GC varied only in the range of the statistical deviation of the two methods. A larger discrepancy was observed for betaine. The values obtained after precipitation of betaine as a reineckate were up to 60% higher than those of the HPLC measurements. The results of the two methods were the same if the samples were filtered through a microporous membrane with a cut-off of 20 kDa. The applicability of the HPLC method for on-line process monitoring was shown in the context of an experiment being run for 40 h.

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 form (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.