Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli.

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28 degrees C, heat shock of the culture from 37 to 42 degrees C and from 37 to 45 degrees C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42 degrees C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.

Ca2+ is a cofactor required for membrane transport and maturation and is a yield-determining factor in high cell density penicillin amidase production.

Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca(2+) per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the pre-pro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat- (E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca(2+) binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca(2+) was targeted to the membrane but not translocated. Influence of Ca(2+) in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca(2+) in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca(2+) content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PA(EC)) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca(2+) or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.

Degradation of polycyclic aromatic hydrocarbons and long chain alkanes at 60-70 degrees C by Thermus and Bacillus spp [corrected].

Although polycyclic aromatic hydrocarbons (PAH) and alkanes are biodegradable at ambient temperature, in some cases low bioavailabilities are the reason 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 degrees C. The microorganisms used grew on hydrocarbons as sole carbon and energy source. Optimal growth temperatures were in the range of 60-70 degrees 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 (1 h)(-1) pyrene and 1000 mg (1 h)(-1) hexadecane at 70 degrees 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.

Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp. A2 degrading phenol at 65 degrees C.

In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp. A2 at 65 degrees C. A high yield coefficient of Y(x/s)=0.84 g cell dry weight g(-1) phenol was measured at a dilution rate of 0.5 h(-1). At the same dilution rate the coefficient for maintenance metabolism (m(s)) was determined to be 0.045 g phenol g(-1) cell dry weight h(-1). The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l(-1) was 0.9 h(-1). Up to 7 g phenol l(-1) per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l(-1)). Additionally, yield coefficients for oxygen and ammonium are reported.

Sedimentation behaviour of sludge particles in a biogas tower reactor and the function of a hydrostatically pressurized sedimenter.

It was found that anaerobic sludge particles contain gas bubbles. Due to the compressibility of the bubbles, which are entrapped in the sludge agglomerates, a pressure-dependent sedimentation characteristic of the sludge particles was found and mathematically described. Enhanced hydrostatic pressure results in a considerable increase of the settling velocity of sludge particles. On this basis the concept of a hydrostatically pressurized sedimenter is presented and its performance analyzed. Due to the hydrostatical pressurization of the sedimenter the produced gas is kept in the liquid phase until the liquid phase is saturated. To avoid a degassing of the liquid which results in the undesired release of gas bubbles a maximum liquid dwell time in the sedimenter must not be exceeded.

Macro- and microscopic in-situ observation of gas bubbles and sludge particles in a biogas tower reactor.

Macroscopic and microscopic in-situ observation of particles and gas bubbles are used to get precise impressions of the hydrodynamical characteristics of a biologically active suspension. Moreover, values of in-situ velocities and particle densities can be gained by using these methods. The suspended anaerobic sludge revealed an extensive fibrous structure ('fur') on its surface. The observed microfibers have a profound influence on the settling/flotation behavior of the particles because they increase the effective particle volume, they may trap gas bubbles and they favor agglomeration. The biomass particles do not appear as single spherical objects but due to its fibrous structure on the outside as strongly interacting mass. The compressibility of the bubbles which are entrapped in the sludge agglomerates results in a pressure-dependent density of the sludge particles.

Effect of glucose, maltose, soluble starch, and CO2 on the growth of the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus was cultivated in batch and continuous fermentations on different carbon substrates. The cultivation of P furiosus on soluble starch as the only carbon source resulted in cell densities three times higher than in cultivations on maltose, 1.06 x 10(10) cells/ml compared to 3.4 x 10(9) cells/ml. The yield coefficient, Y(x/y) = 0.12 g/g, and the growth rate, mu = 0.33 h(-1), were almost equal on soluble starch and on maltose, but on glucose no growth could be detected. An inhibitory effect of glucose, when added to other carbon substrates, also could not be found. Isobutyric and isovaleric acid were detected as novel metabolites produced by P. furiosus. Inhibitory effects of these acids, as well as of the well-known products acetic acid, propionic acid, and alanine, could be precluded. Concentrations of 10% CO2 in the gas supply respective to the exhaust gas enhanced the growth of P furiosus significantly. The maximum cell number was two orders of magnitude higher than was observed with pure nitrogen. Further increase of the CO2 concentration up to 100% had no significant effect on the growth of P. furiosus.