New biotechnological approaches for the development of a fluorescence-based multiarray biosensor for environmental agrifood monitoring and for the production of algal biomass
Author(s)
Silletti, Silvia
Date Issued
May 30, 2014
Type
Doctoral Thesis
Abstract
Thesis work is composed of two parts both realized for two independent industrial projects.
In the first part the green microalga Chlorella minutissima was used to produce biomass at low costs for applications in biofuel production. Biofuel generation requires the production of lipids from biomass at low costs. In order to produce biomass, the green alga Chlorella minutissima was chosen because in literature is reported to produce large amounts of lipids. It is known that several algae of Chlorella genus are able to establish endosymbiosis with other organisms and the most famous example of this association is the endosymbiosis between Chlorella vulgaris and Paramecium Bursaria.
During the experiments focused on the selection of the best culture medium deriving from farm and industrial wastes, we isolated two protists which are able to establish endosymbiosis with Chlorella minutissima. The two protists were identified as Tetrahymena pyriformis and Chilomonas paramecium. We found that the presence of these organisms in algal cultures determines an increase of the biomass/lipid production and a longer vitality of the mixed culture compared to culture with algae alone. An international patent was deposited.
In order to identify with precision the symbiotic organisms, molecular analyses were attempted. In particular, we used PCR analyses on total DNA extracted from our isolated microorganisms and the same organisms obtained from culture collections, but no results were obtained.
The second part of the thesis, divided in two subparts, is focused on the development of a multiarray biosensor based on fluorescence for environmental and agrifood monitoring. Biosensors are defined as devices which use a biological recognition element retained in direct spatial contact with transduction system.
In the part IIa of this thesis, we studied symbiosis between Chlorella minutissima and protists in order to use them as biomediators.
The symbiosis establishment determines changes in the culture that produces aggregates of different size in relation to the culture age. The algae are surrounded of the thick membrane called PV (perialgal vacuole), being more resistant to environmental stresses. We studied why some algae are able to establish symbiosis and others not. In literature various hypotheses were formulated, but no evident results were obtained. We hypnotized that this phenomenon could be determined by the presence of some particular lipids. We performed lipid analyses on samples of two symbiotic algae (Chlorella minutissima and sorokiniana H1986) and two non symbiontic ones (Chlorella prototecoides and mirabilis) that indicated that there is a great difference in the total lipid content
between the two algal classes; however, no differences in the fatty acid content could be related to the different behavior.
Single culture of Chlorella minutissima and mixed culture of Chlorella minutissima and protists were tested as biomediators in fluorescence-based biosensor in order to detect photosynthetic herbicides. The experiments have shown that symbiotic culture is able to reveal presence of the analyte diuron at the low concentrations of 10-10M and that the response is higher than that obtained with single Chlorella cultures.
In the last part of this work, we worked to develop a multiarray biosensor for milk safety and quality assessment. For the analysis of safety, we have chosen three different analytes: diuron (a photosynthetic herbicide), chlorpyrifos (an organophosphorous pesticide) and catechol (a phenolic compound). As biomediators, we used whole cells of Chlamydomonas, the enzyme acetylcholinesterase from Electrophorus electricus and the enzyme tyrosinase from mushroom, respectively. For milk quality monitoring, we revealed urea, lactose and L-lactic acid, using as biomediators urease from Canavalia ensiformis, β-galactosidase from Aspergillus oryzae and L-lactate dehydrogenase from Lactobacillus leichmannii, respectively. Enzyme fluorescence activity was guaranteed by the use of FITC (Fluorescein 5(6)-isothiocyanate) and 5(6)-Carboxynaphthofluorescein. Measurements were performed in buffer and subsequently in milk. Thanks to the identification of the optimal measurement conditions for each biomediator, it was possible to develop in collaboration with Biosensor s.r.l. a multiarray biosensor with six detection cells that permit simultaneous milk on line analyses directly in cowshed.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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