Thomas Maskow

Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium

ABSTRACT The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter−1 ectoine with a space-time yield of 40 mg liter−1 h−1, with the vast majority of the ectoine being excreted.

A combination method to study microbial communities and activities in zinc contaminated soil.

Zinc (Zn) plays a special role in soil ecology and fertility because it can support the growth of soil organisms or inhibit their growth depending on its concentrations. In this work, the effects of different concentrations of Zn on soil microbial communities and activities were analyzed by loading five different doses of Zn (160-6000 microg g(-1)) into a wheat surface soil. The microbial metabolic process revealed a significant bimodal pattern at high concentrations of Zn (>1920 microg g(-1)). This phenomenon suggested that soil microorganisms were very sensitive to zincous poisoning. A variety of soil quality properties were also measured and assessed. The results showed slower bacterial growth in soil cultures polluted with high levels of Zn. In addition, two kinds of fungi were identified by morphology and glomalin-related soil protein content in the Zn-contaminated soil. The growth of the first kind was inhibited with increase in Zn concentration. By contrast, the second kind could survive and continue to grow with increasing doses of Zn at 160-1920 microg g(-1) and its growth began to decline with further increase in Zn concentration. Finally, the fungus could not survive at very high (6000 microg g(-1)) Zn concentration. In this work, we conclude that soil microbial communities and activities can adapt to Zn pollution to a certain extent.

Calorimetrically recognized maximum yield of poly-3-hydroxybutyrate (PHB) continuously synthesized from toxic substrates

The broader usage of poly-beta-hydroxybutyrate (PHB), for instance as bulk plastics, calls for cheap raw materials and greater overall process efficiency. The bacterial synthesis is generally induced and promoted by the limitation of growth via nitrogen, oxygen or phosphate depletion with the simultaneous excess and higher concentration of the carbon substrate. Consequently, toxic substrates have been considered unsuitable for PHB synthesis. Nevertheless, a single-stage continuous process for producing PHB from toxic substrates using microorganisms was developed and is reported here. The maximum heat flux during continuous growth and the maximum yield of PHB versus the substrate consumption rate were found to coincide. This suggests the possibility of controlling the conversion of a growth-inhibiting substrate into PHB and maximizing the process efficiency. The observed correlation occurred irrespective of the substrates investigated (phenol or sodium benzoate), the PHB-producing strain (Ralstonia eutropha JMP 134 or Variovorax paradoxus JMP 116), or the type of limitation imposed. The maximum PHB yields obtained comprised up to 50% of cell dry mass.

Chip calorimetry for fast and reliable evaluation of bactericidal and bacteriostatic treatments of biofilms.

ABSTRACT Chip calorimetry is introduced as a new monitoring tool that provides real-time information about the physiological state of biofilms. Its potential for use for the study of the effects of antibiotics and other biocides was tested. Established Pseudomonas putida biofilms were exposed to substances known to cause toxicity by different mechanisms and to provoke different responses of defense and resistance. The effects of these compounds on heat production rates were monitored and compared with the effects of these compounds on the numbers of CFU and intracellular ATP contents. The real-time monitoring potential of chip calorimetry was successfully demonstrated by using as examples the fast-acting poisons formaldehyde and 2,4-dinitrophenol (DNP). A dosage of antibiotics initially increased the heat production rate. This was discussed as being the effect of energy-dependent resistance mechanisms (e.g., export and/or transformation of the antibiotic). The subsequent reduction in the heat production rate was attributed to the loss of activity and the death of the biofilm bacteria. The shapes of the death curves were in agreement with the assumed variation in the levels of exposure of cells within the multilayer biofilms. The new monitoring tool provides fast, quantitative, and mechanistic insights into the acute and chronic effects of a compound on biofilm activity while requiring only minute quantities of the biocide.

Observation of non-linear biomass-capacitance correlations: reasons and implications for bioprocess control.

Electrical capacitance has been discussed as a real time measure for living biomass concentration in technical bioreactors such as brewery (fermentation) tanks. Commonly, a linear correlation between biomass concentration and capacitance is assumed. While following the growth and subsequent lipid formation of the yeast Arxula adeninivorans we observed non-linearity between biomass concentration and capacitance. Capacitance deviation from linearity coincided with incipient lipid formation and depended on the intracellular lipid content. As the extent of deviation between capacitance and biomass concentration was proportional to the lipid concentration, it was considered as a quantitative measure of intracellular product formation. The correlation between shifts in dielectric relaxation (summarized as characteristic frequency of the Cole-Cole equation) and lipid content could not be explained by interfacial polarization on the lipid droplets alone. However, the parameters of the Cole-Cole equation were found to be a clear indicator for different phases of growth and lipid production. Integrating all results in a redundancy analysis (RDA), we were able to accurately describe the formation of cellular lipid inclusions. Our measurements are thus potentially valuable as components of future bioprocess control strategies targeting intracellular products such as proteins or biopolyesters.

On-line monitoring of lipid storage in yeasts using impedance spectroscopy

Bioremediation technologies and many environmentally sound biosyntheses rely on the catalytic potential of whole cells. For analyzing and controlling such processes robust real-time indicators for the concentration of intact cells such as impedance are required. The conventional method measures the capacitances of cell suspensions at one or two frequencies and correlates them with biomass concentrations. However, cell inclusions such as lipid droplets or overproduced enzymes may block intracellular ion paths, thereby possibly modifying the dielectric properties of the cells. To test the hypothesis that the total impedance spectrum into the analysis may provide useful information about cell inclusions, the impedance spectrum of a technical culture of the oleaginous yeast Arxula adeninivorans was measured and evaluated every 15s. This yeast is a good test object since it stores the excess of assimilated carbon in experimentally controllable lipid droplets. Upon correction for possible impedance signal interferences, we derived different empirical methods suitable to indicate incipient lipid formation. The methods were designed to act on-line and are thus principally suited for real-time monitoring of cell inclusions. In search for optimised bioprocess monitoring we tested a heuristic spectrum analysis using integrative statistics (RDA). With this approach we were able to accurately detect the formation of cell inclusions, which is potentially valuable for future bioprocess control strategies.

Potentials and limitations of miniaturized calorimeters for bioprocess monitoring.

In theory, heat production rates are very well suited for analysing and controlling bioprocesses on different scales from a few nanolitres up to many cubic metres. Any bioconversion is accompanied by a production (exothermic) or consumption (endothermic) of heat. The heat is tightly connected with the stoichiometry of the bioprocess via the law of Hess, and its rate is connected to the kinetics of the process. Heat signals provide real-time information of bioprocesses. The combination of heat measurements with respirometry is theoretically suited for the quantification of the coupling between catabolic and anabolic reactions. Heat measurements have also practical advantages. Unlike most other biochemical sensors, thermal transducers can be mounted in a protected way that prevents fouling, thereby minimizing response drifts. Finally, calorimetry works in optically opaque solutions and does not require labelling or reactants. It is surprising to see that despite all these advantages, calorimetry has rarely been applied to monitor and control bioprocesses with intact cells in the laboratory, industrial bioreactors or ecosystems. This review article analyses the reasons for this omission, discusses the additional information calorimetry can provide in comparison with respirometry and presents miniaturization as a potential way to overcome some inherent weaknesses of conventional calorimetry. It will be discussed for which sample types and scientific question miniaturized calorimeter can be advantageously applied. A few examples from different fields of microbiological and biotechnological research will illustrate the potentials and limitations of chip calorimetry. Finally, the future of chip calorimetry is addressed in an outlook.

Evaluation of solvent tolerance of microorganisms by microcalorimetry

Solvent tolerance is important because it allows microorganisms to grow at high concentrations of organic solvents. Organisms capable of surviving under these extreme conditions have great application in bioremediation of contaminated sites. In our study, four strains of Pseudomonas sp. B1 and J2, Acinetobacter sp. B2 and J6 which were isolated from the activated sludge were used to evaluate the solvent tolerance by microcalorimetry. The strains B2 and J2 showed high tolerance to organic solvent as they could grow well in the medium containing 10 vol% of benzene and 70 vol% of toluene, respectively. The higher the growth rate constant, the higher the solvent tolerance of the strains isolated. The microbial growths obtained by microcalorimetry were in good agreement with the results determined by OD(600). The decrease in growth rate constant and the change in total thermal effect in the presence of organic solvents were in agreement with the data reported in the literatures. Strains B1 and B2 degraded about 67% and 94% of 0.1 vol% benzene within 72 h in a medium with benzene as the sole carbon source, respectively. Strains J2 and J6 degraded approximately 92% and 85% of 0.1 vol% toluene within 72 h, respectively. The related degradation genes detected in previous study in these strains highlight an important potential use of those bacteria for the clean-up of benzene and toluene in the environment.

Thermokinetic description of anaerobic growth of Halomonas halodenitrificans using a static microcalorimetric ampoule technique

Efficiency and velocity of growth are key variables to consider when designing any microbial biotechnological process. Selection of the optimal strain and description of environmental effects on growth patterns require rapid information about relevant parameters. Calorimetry is particularly suitable for providing such data, provided it can simultaneously perform many measurements and the apparatus is as simple as possible. The simplest experimental set-up measures the heat flux of microorganisms growing in a static, sealed ampoule. But, how reliable and reproducible are the growth rates and growth yield coefficients obtained from such a system? To answer this question, the strain Halomonas halodenitrificans CCM 286(T) was grown on glycerol with nitrate as the terminal electron acceptor in a multi-channel isothermal heat conduction calorimeter in such a way that growth was predominantly influenced by availability of the oxidant. The time course of the heat fluxes up to the maximum attained was successfully modelled using integrated Monod kinetics. The reproducibility of the specific growth rate obtained was excellent (standard deviation less than 1% for a single measurement and less than 3% for a couple of measurements) and agreed well with figures reported in the literature. An Arrhenius-type model, consisting of one term for the activation and one for the inactivation of the microbial catalyst, was found to fit the whole specific growth rate versus temperature curve.

Investigation of the toxic effect of cadmium on Candida humicola and Bacillus subtilis using a microcalorimetric method.

In this study, the technique of microcalorimetry based on heat-output by aerobic bacterial respiration was explored to evaluate the toxic effect of cadmium on Candida humicola, Bacillus subtilis, singularly or in a mixture of both. Power-time curves of the growth metabolism of C. humicola and B. subtilis and the effect of Cd(2+) were studied using the TAM III (the third generation thermal activity monitor) multi-channel microcalorimetric system, isothermal mode, at 28 degrees C. The differences in shape of the power-time curves and the thermodynamic and kinetic characteristics of microorganisms growth were compared. The effect of cadmium added into microorganism would significantly reduce the life cycle and change the thermal effect of microbial metabolic process with different concentrations of Cd(2+). The experimental results revealed that at the same concentration, the sequence of inhibitory ratio (I) and maximum thermal power (P(max)) of the Cd(2+) was: mixed microorganisms>C. humicola>B. subtilis. The sequence of total thermal effect (Q(total)) and growth rate constant (k) is mixed microorganisms>B. subtilis>C. humicola. These results are important to further studies of the physiology and pharmacology of C. humicola and B. subtilis and may support the theory of restoring contaminated soil.