APPLICATION OF ISOTHERMAL CALORIMETRY TO MONITOR MICROBIAL GROWTH AND METABOLISM IN SOILS AND FRESH FOOD PRODUCTS

This PhD research project aims to verify the use of Isothermal Calorimetry (IC) as a valid technique to assess the microbial growth in different environments, in either liquid or solid state. In liquid stirred cultures every bacterium grows essentially in the same environmental conditions, and this simplifies significantly the study and characterization of the growth processes, while bacterial growth in solid media is much more difficult to study. Isothermal calorimetry (IC) represents a powerful method to directly monitor the biological activity of such living systems since it provides direct quantitative information about the relevant energetics and kinetics. In the first part of the research, we focused our attention on the study of two microorganisms involved in the oxidation/reduction of arsenic which is a ubiquitous contaminant related to natural weathering and industrial or agricultural activities and strongly affects the quality of cultivable soils used for the production of edible crops. In this part of the study, calorimetric measurements are proven to be suitable to evaluate the toxic effect of arsenic on the growth of two microbial strains (Achromobacter sp. and As5-13 Bacillus sp. 3.2) isolated from soil and groundwater. The toxicity and mobility of this element depend on chemical speciation and its oxidation state, and are specific for each microorganism, for instance inorganic arsenite As(III) is more toxic and mobile than arsenate As(V). Microorganisms can directly affect the redox balance As(V)/As(III) shifting it toward the species that can be more easily processed and/or absorbed by scavenger plants. IC was applied in the first step to assess the effects of arsenic on the growth, metabolism and the As(V)/As(III) redox capability of Achromobacter sp. As5-13 strain, a well characterized microorganism isolated from an agricultural soil. Other parallel measurements were performed with conventional methods, such as turbidity or microbial count, and standard chemical analysis to determine the arsenic concentration. In the second step IC and classical microbiological methods were applied to evaluate the influence of As(V) and As(III) on the growth and metabolism of Bacillus sp. 3.2 (isolated from groundwater) in different culture medium, namely, Tris Mineral Medium with Gluconate (TMMG) with the addition of yeast extract, and Tryptone Soya Broth (TSB). The IC thermo-grams in combination with the microbiological counts and the chemical analysis data on the arsenic oxidation status during the microbial activity, permitted to discriminate the influence of arsenic on the energetic aspects of the microbial metabolism in conditions that mimic different kind of cultivable soils. Regarding the second part of the research, we started exploring bacterial growth in solid state media choosing two different foods matrixes: carrots and corn salad. In the first step we used IC to investigate the effect of different cuts on carrots (cylinders, sticks and juliennes) in order to monitor the kinetics of microbial growth and to simplify this research we selected juice carrot and Pseudomonas fluorescens sp., a well characterized microorganism isolated from carrots, as a model microorganism. In the second step, IC studies were performed to assess the shelf life of corn salad, choosing three different temperatures of storage (5, 10 and 20°C), studying the kinetic evolution of chemico-physical (color, total phenols, moisture, pH variation and flow heat) and microbiological indices. We used for this study two different calorimeters in order to evaluate the influence of the temperature on the kinetics of microbial growth. The results obtained in this project showed that calorimetric method used in combination with other relevant methods is a very powerful tool in studying the kinetic and metabolic growth of bacteria in different fields. Moreover, Calorimetry assays, essentially consisting in on-line measurements, are very precise and easy to carry out in comparison with other methods currently used.