Halogenated aliphatic and aromatic aerobic biodegradation via direct metabolism and cometabolism: batch and continuous tests

In this thesis the application of biotechnological processes based on microbial metabolic degradation of halogenated compound has been investigated. Several studies showed that most of these pollutants can be biodegraded by single bacterial strains or mixed microbial population via aerobic direct metabolism or cometabolism using as a growth substrates aromatic or aliphatic hydrocarbons. The enhancement of two specific processes has been here object of study in relation with its own respective scenario described as follow: 1st) the bioremediation via aerobic cometabolism of soil contaminated by a high chlorinated compound using a mixed microbial population and the selection and isolation of consortium specific for the compound. 2nd) the implementation of a treatment technology based on direct metabolism of two pure strains at the exact point source of emission, preventing dilution and contamination of large volumes of waste fluids polluted by several halogenated compound minimizing the environmental impact. In order to verify the effect of these two new biotechnological application to remove halogenated compound and purpose them as a more efficient alternative continuous and batch tests have been set up in the experimental part of this thesis. Results obtained from the continuous tests in the second scenario have been supported by microbial analysis via Fluorescence in situ Hybridisation (FISH) and by a mathematical model of the system. The results showed that both process in its own respective scenario offer an effective solutions for the biological treatment of chlorinate compound pollution.

Effetto della temperatura di conservazione del latte, dalla raccolta all'affioramento, sulla microflora del latte, del sieroinnesto e del formaggio Trentingrana

This PhD research is part of a project addressed to improve the quality of Grana Trentino production. The objectives were to evaluated if milk storage and collection procedures may affect cheese-making technology and quality. Actually the milk is collected and delivered to the cheese factory just after milking in 50 L cans without refrigeration or in tanks cooled at 18 °C. This procedure is expensive (two deliveries each day) and the milk quality is difficult to preserve as temperatures are not controlled. The milk refrigeration at the farm could allow a single delivery to the dairy. Therefore it could be a good strategy to preserve raw milk quality and reduce cheese spoilage. This operation may, however, have the drawbacks of favouring the growth of psychrotrophic bacteria and changing the aptitude of milk to coagulation. With the aim of studying the effect on milk and cheese of traditional and new refrigerated technologies of milk storage, two different collection and creaming technologies were compared. The trials were replicated in three cheese factories manufacturing Grana Trentino. Every cheese-making day, about 1000 milk liters were collected from always the same two farms in the different collection procedures (single or double). Milk was processed to produce 2 wheels of Grana trentino every day. During the refrigerated trials, milk was collected and stored at the farm in a mixed tank at 12 or 8 °C and then was carried to the dairy in truck once a day. 112 cheese making day were followed: 56 for traditional technology and 56 for the refrigerated one. Each one of these two thechnologies lead to different ways of creaming: long time in the traditional one and shorter in the new one. For every cheese making day we recorded time, temperatures and pH during the milk processing to cheese. Whole milk before ceraming, cream and skim milk after creaming, vat milk and whey were sampled during every cheese-making day for analysis. After 18 months ripening we opened 46 cheese wheels for further chemical and microbiological analyses. The trials were performed with the aim of: 1 estimate the effect of storage temperatures on microbial communities, physico-chemical or/and rheological differences of milk and skim milk after creaming. 2 detect by culture dependent (plate counts) and indipendent (DGGE) methodolgies the microbial species present in whole, skimmed milk, cream and cheese sampled under the rind and in the core; 3 estimate the physico-chemical characteristics, the proteolytic activity, the content of free aminoacids and volatile compounds in 18 months ripened Grana Trentino cheeses from different storing and creaming of milk technologies. The results presented are remarkable since this is the first in-deep study presenting microbiological and chemical analysis of Grana Trentino that even if belonging to Grana Padano Consortium, it is clearly different in the milk and in the manufacturing technology.

Bioremediation of PCB-contaminated marine sediments: From identification of indigenous dehalorespirers to enhancement of microbial reductive dechlorination

Marine sediments are the main accumulation reservoir of organic recalcitrant pollutants such as polychlorinated biphenyls (PCBs). In the anoxic conditions typical of these sediments, anaerobic bacteria of the phylum Chloroflexi are able to attack these compounds in a process called microbial reductive dechlorination. Such activity and members of this phylum were detected in PCB-impacted sediments of the Venice Lagoon. The aim of this work was to investigate microbial reductive dechlorination and design bioremediation approaches for marine sediments of the area. Three out of six sediment cultures from different sampling areas exhibited dechlorination activities in the same conditions of the site and two phylotypes (VLD-1 and VLD-2) were detected and correlated to this metabolism. Biostimulation was tested on enriched dechlorinating sediment cultures from the same site using five different electron donors, of which lactate was the best biostimulating agent; complementation of microbial and chemical dechlorination catalyzed by biogenic zerovalent Pd nanoparticles was not effective due to sulfide poisoning of the catalyst. A new biosurfactant-producing strain of Shewanella frigidimarina was concomitantly obtained from hydrocarbon-degrading marine cultures and selected because of the low toxicity of its product. All these findings were then exploited to develop bioremediation lab-scale tests in shaken reactors and static microcosms on real sediments and water of the Venice lagoon, testing i) a bioaugmentation approach, with a selected enriched sediment culture from the same area, ii) a biostimulation approach with lactate as electron donor, iii) a bioavailability enhancement with the supplementation of the newly-discovered biosurfactant, and iv) all possible combinations of the afore-mentioned approaches. The best bioremediation approach resulted to be a combination of bioaugmentation and bioremediation and it could be a starting point to design bioremediation process for actual marine sediments of the Venice Lagoon area.

Evaluation of 3D cell culture systems for host-pathogen interaction studies

Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.