Chip-calorimetry provides real time insights into the inactivation of biofilms by predatory bacteria.

Control or removal of undesired biofilms has frequently been found to be quite difficult. In addition to biocidal or antibiotic chemicals or materials designed to prevent biofouling, biological control agents appear to be promising. Reports of bacterial predators eradicating biofilms or eliminating pathogens motivate a more systematic screening of biofilm-eliminating bacterial predators. Unfortunately, the analysis of the eradication process is demanding. In the present study, chip-calorimetry was applied to monitor the elimination of Pseudomonas sp. biofilms by Bdellovibrio bacteriovorus. The method uses metabolic heat as a real-time parameter for biofilm activity. The method is non-invasive, fast and convenient due to real-time data acquisition. In addition, heat-production data can reveal information about the energetics of the predator-prey interaction. The calorimetric results were validated by confocal laser scanning microscopy. The approach described may be useful for the screening of biofilm susceptibility to different predators.

Isothermal titration calorimetry - a new method for the quantification of microbial degradation of trace pollutants.

The environmental fate and, in particular, biodegradation rates of hydrophobic organic compounds (HOC) are of high interest due to the ubiquity, persistence, and potential health effects of these compounds. HOC tend to interact with bioreactor materials and sampling devices and are frequently volatile, so that conventionally derived degradation parameters are often biased. We report on the development and validation of a novel calorimetric approach that serves to gain real time information on the kinetics and the physiology of HOC bioconversion in aqueous systems while overcoming weaknesses of conventional biodegradation experiments. Soil bacteria Mycobacterium frederiksbergense LB501T, Rhodococcus erythropolis K2-3 and Pseudomonas putida G7 were exposed to pulsed titrations of dissolved anthracene, 4-(2,4-dichlorophenoxy)butyric acid or naphthalene, respectively, and the thermal responses were monitored. The combinations of strains and pollutants were selected as examples for complete and partial biodegradation and complete degradation with storage product formation, respectively. Heat production signals were interpreted thermodynamically and in terms of Michaelis-Menten kinetics. The half-saturation constant k(D) and the degradation rate r(D)(Max) were derived. Comparison with conventional methods shows the suitability to extract kinetic degradation parameters of organic trace pollutants from simple ITC experiments, while thermodynamic interpretation provided further information about the metabolic fate of HOC compounds.

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

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.

Miniaturized calorimetry - a new method for real-time biofilm activity analysis.

The partial dissipation of Gibbs energy as heat reflects the metabolic dynamic of biofilms in real time and may also allow quantitative conclusions about the chemical composition of the biofilm via Hess' law. Presently, the potential information content of heat is hardly exploited due to the low flexibility, the low throughput and the high price of conventional calorimeters. In order to overcome the limitations of conventional calorimetry a miniaturized calorimeter for biofilm investigations has been evaluated. Using four thermopiles a heat production with spatial and temporal resolutions of 2.5 cm(-1) and 2 s(-1) could be determined. The limit of detection of the heat flow measurement was 20 nW, which corresponds to the cell density of an early stage biofilm (approx. 3x10(5) cells cm(-2)). By separating biofilm cultivation from the actual heat measurement, a high flexibility and a much higher throughput was achieved if compared with conventional calorimeters. The approach suggested allows cultivation of biofilms in places of interest such as technological settings as well as in nature followed by highly efficient measurements in the laboratory. Functionality of the miniaturized calorimeter was supported by parallel measurements with confocal laser scanning microscopy and a fiber optic based oxygen sensor using the oxycaloric equivalent (-460 kJ mol-O2(-1)).

Calorimetrically obtained information about the efficiency of ectoine synthesis from glucose in Halomonas elongata.

Compatible solutes are becoming more and more attractive commercially. Thus, knowledge of the efficiency of synthesis of compatible solutes from different carbon substrates is very important. As the growth rate and rates of formation of compatible solutes correspond to the heat flux, calorimetric measurements are particularly suitable for providing this information. By growing microorganisms continuously in a calorimeter, and generating a feeding stream with gradually increasing salinity without changing any other growth conditions, we were able to determine the efficiency of growth-associated synthesis of compatible solutes. This was shown for Halomonas elongata DMSZ 2581(T) growing on glucose, which synthesizes (at 25 degrees C) 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) as its main osmotic counterweight. The requirement of biologically usable energy for its growth-associated synthesis was found to be very low: a 100% efficiency of the conversion of the substrate-carbon into ectoine is both theoretically possible and was reached approximately in practice. The growth rate and yield coefficient were essentially independent of the ectoine formation rate, and the rate of substrate-carbon assimilation was far greater than the rate of dissimilation. The specific maximum growth rate was limited by the rate of formation of ectoine.

A calorimetrically based method to convert toxic compounds into poly-3-hydroxybutyrate and to determine the efficiency and velocity of conversion.

A fed-batch method for converting toxic substrates into poly-3-hydroxybutyrate is presented. The method involves a series of batch-growth processes, regulated by adding small amounts of carbon substrate, during the course of which the concentration of the nitrogen source decreases and controls the distribution of the substrate-carbon assimilated. The addition of carbon substrate is controlled, and the small changes that occur in the growth pattern are interpreted using high-resolution reaction calorimetry. The method was tested with Ralstonia eutropha DSM 4058 growing on phenol, and Variovorax paradoxus DSM 4065 growing on sodium benzoate. The maximum carbon conversion efficiencies (CCEs) obtained, 23% and 27% respectively, were compared with the theoretically possible values.

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.