Surface Active Compounds by Gordonia and their applications in environmental remediation

This chapter reports the outline of the results presented in the next chapters. CHAPTER 2 deals with the isolation and the characterization of new SAC-producing strains. From a site, chronically contaminated by diesel fuel, three bacterial strains (M22, BS25 and BS29) belonging to the Gordonia genus were isolated. Results showed that those Gordonia strains were able to grow using a wide range of straight and branched aliphatic hydrocarbons as carbon and energy source and to produce at least two classes of surface-active compounds. Emulsifying agent(s) was released in the culture medium when bacteria grew both on hydrocarbons and water-soluble substrates. Cell-bound biosurfactant(s), that reduce the surface tension, was produced on hydrocarbons; however, its production was significantly lower on water soluble substrates. Solvent extraction and Thin Layer Chromatography analyses allowed us to classify the cell-bound biosurfactants as glycolipids and to confirm that the chemical nature of the extracellular bioemulsans is different. The relation among growth phase, surface-active compound production and cell-surface properties was analysed in kinetic experiments on hydrocarbons. Gordonia sp. BS29 synthesised, and released extracellularly, bioemulsan(s) during the exponential phase with n-hexadecane as carbon and energy source. The production of biosurfactant(s) started in the exponential phase and its concentration increased during the following linear growth. Furthermore, the adhesion of bacterial cells to hydrocarbons decreased during growth. Our results let to hypothesize a change in the mode by which Gordonia cells access the substrate during growth on hydrocarbons. The results reported in CHAPTER 3 deals with the evaluation of the application potentialities of the isolates and the biosynthesised SACs in environmental restoration technologies. Microbial amphiphiles and their producing strains can be use both as enhancers of biodegradation rate in bioremediation technologies of hydrocarbon contaminated soil and chemico-physical technologies of soil washing. The applicability of our strains and SACs were evaluated for the following applications: bioremediation of (i) aliphatic and (ii) aromatic hydrocarbon contaminated soil and (iii) washing of soil contaminated by crude oil. Microcosm bioremediation experiments were carried out and the effect of BS29 and M22 bioaugmentation and the bioemulsans on biodegradation rate and extent were evaluated also in comparison to the effect of the rhamnolipids, biosurfactants known for their capacities of enhancing bioremediation. Over time, the humidity was kept constant (25%) and the analyses of residual hydrocarbons, total microorganisms and hydrocarbon-degrading microorganisms were carried out. Hydrocarbon biodegradation data were analysed by Analysis of Variance (ANOVA) to compare the tested conditions with the control without any addition. The dependent variables for the comparison were the biodegradation rate and the residual concentration of each contaminant at the end of the experiments. The bioremediation results showed that the bioemulsan is able to effectively reduce final concentration of recalcitrant branched hydrocarbons even not at the same extent of rhamnolipids. Batch soil washing experiments were carried and we compared the effect of the BS29 bioemulsans with the ones of the sole water and a mixture of rhamnolipids (biosurfactant already experimented and used in soil washing techniques) on the removal of the pollutants from soil. Experimental Design Techniques were utilised for an efficient experiment planning and to evaluate the effect of the operational parameters on pollutants removal. The investigated factors were (i) the time of washing and (ii) the ratio between the volume of washing solution and the weight of soil. The use of a crude solution of the BS29 bioemulsans in different conditions resulted in a mean of crude oil removal of 43.1%, while the mean removal in rhamnolipid experiments was 50.8%. ANOVA test showed that both of them are significantly different (α=0.05) from the mean of sole water removal (19.5%). Furthermore, in bioemulsan experiments, a significant effect of time of washing was observed with a positive correlation with the extent of the oil removal. On the contrary, in the tested experimental conditions, neither the solution:soil ratio nor the time of washing significantly affects the removal in rhamnolipids experiments. The results obtained from the optimisation procedure for the maximisation of biosurfactant concentration in cultural broth are reported in CHAPTER 4. The optimisation process involved three consecutive steps. In the first step a two level 2(8-2) Fractional Factorial Design (FFD) was used to identify cultural factors that have a significant influence on biosurfactant(s) biosynthesis. Concentrations of inorganic phosphorous and sodium chloride were found as most important factors affecting yield of biosurfactant biosynthesis Then, on the selected factors, a steepest ascent procedure and a Central Composite Design (CCD) were applied to obtain a second order polynomial function which fitted the experimental data in the vicinity of the optimum. The factors taken into account were inorganic nutrients, such as phosphorous, ammonium and micronutrients, the carbon sources and the time of growth. With the optimised broth composition we obtained a more than 16-fold increase in the biosurfactant concentration compared to the normal BH broth, reaching a Critical Micelle Diluton (CMD) value (129.43 (95% confidence interval: 119.47 – 139.40)) among the highest in literature. In CHAPTER 5 the whole conclusions of the project are drawn and the perspectives for the future are put forward. CHAPTER 6 presents the results of the studies dealing with the ecology and the survival of thermophilic bacteria in cold soil environments. Population analysis of thermophiles in rainwater and air samples has shown distinct differences supporting the view that long distance global transport and deposition in rainwater are a possible source of replenishment of the soil thermophile populations.