Titolo della tesi: Selection and characterization of microalgae strains able to grow on alternative carbon substrates for industrial application, with an outlook on strain improvement
Microalgae, a phylogenetically diverse group of photosynthetic eukaryotes, are able to produce a wide range of compounds of industrial interest. These metabolites are relevant for the food, feed, nutraceutical, and pharmaceutical industries. They may as well be used in green technologies, for the production of bioplastics and biofuels. Even though commercial production of microalgae has a history of approximately 60 years, the focus has been only on a handful of species. The number of industrial species is very low considering the vast number of species recognized and the estimated number of algae yet to be identified. Selecting and characterizing new strains of industrial value may therefore be of interest, which may expand the horizon of commercial cultivation. This could be achieved through bioprospecting, focusing on key traits significant to reach economically viable production.
In the first part of the thesis we focused on selecting and characterizing microalgae strains isolated from different areas of Southern California, and strains originated from algal culture collections. The selection criteria were: (1) The ability of a strain to grow in mixotrophic/heterotrophic regime on cheap organic carbon substrate, i.e. corn stover hydrolysate. Identifying a strain capable to grow in these regimes would give versatility to the production process design, in addition to the inherent advantages of these cultivation modes. (2) Ability of a strain to produce metabolites of industrial relevance. (3) Possibility of the strain to be genetically transformed, opening the door to future strain improvement through genetic engineering. Out of the 46 strains screened on the main monosaccharide constituents of corn stover hydrolysate, glucose (Glc) and xylose (Xyl), we identified 25 able to metabolize Glc as a single organic carbon source, of which three strains were able to assimilate Xyl, but only from the complex biomass hydrolysate. All 25 strains capable to utilize Glc could also grow on corn stover hydrolysate. We then focused our attention on two strains able to metabolize Xyl, isolated from the desertic Imperial Valley, California, and identified as Chlorococcum sp., and Desmodesmus sp. Based on information available in the literature on the ability of these strains to produce metabolites of interest they are promising candidates for industrial production. We then tested Chlorococcum sp. and Desmodesmus sp. for genetic transformation and heterologous reporter protein expression, and found the former to be transformable by the applied protocol. Sequencing of the strain may be necessary to improve transformation efficiency as it would allow to identify endogenous regulatory elements for improved vector design.
In the second part of the thesis we focused our attention on a strain able to metabolize lactose, and grow on dairy wastewater. Dairy industry, within the food industry, is amongst the areas that generate the largest volume of waste. Dairy wastewaters are variable in composition, however in general they are characterized by a high load of organic matter, primarily attributed to lactose. Biological treatment approaches are often preferred, where the pollutants in the waste serve as nutrients for microorganisms, primarily fungi and bacteria. Biological water treatment may be coupled with the production of compounds with industrial value. Utilizing algae for biological water treatment is quite promising, as shown by published research; nevertheless, to date only a few species have been shown to metabolize lactose, a necessary trait to reach high water treatment efficiency. The strains identified to be able to grow on Glc in the first part of the thesis were screened on lactose, and on its other monosaccharide constituent apart from Glc, i.e. galactose (Gal). We observed that, of the screened strains, an isolate from the University of California field station, identified as Parachlorella kessleri, was able to metabolize lactose. To the best of our knowledge this is the first report showing this species able to utilize the disaccharide. We then evaluated the growth kinetics of this strain on lactose and on its constituents, Glc and Gal, and on skimmed buttermilk, a waste product originated from the dairy factory Capurso Azienda Casearia S.r.l., in mixotrophic and heterotrophic conditions, and confirmed the ability of the alga to grow on all these substrates, while comparing the biomass productivities and maximum biomass densities on the various substrates.
In the third part of the thesis, we moved in a somewhat different direction, since we put our attention on one of the most relevant industrial species, Chlorella vulgaris, with the aim to contribute to the public knowledge with more information on a trait which may help to optimize the species’ commercial production. C. vulgaris has a robust cell wall, which leads to elevated production cost due to increased energy demands for metabolite extraction, and limits strain improvement by genetic engineering. To date there is limited knowledge on the cell wall composition of this species and on the genetic determinants of its biosynthesis and assembly. In order to obtain novel C. vulgaris with weakened cell wall, a mutagenized population was recently screened for mutants with increased permeability to a fluorescent dye in Prof. Roberto Bassi’s laboratory (University of Verona). We studied the cell wall biochemical composition of the mutants in relation to the wild type with the aim to evaluate which structural components affect the cell wall mechanical properties of C. vulgaris. The biochemical analysis was performed by stepwise extraction of the cell wall polysaccharides with increasingly strong reagents, with subsequent hydrolysis of the polymers to their constituents, and quantification of the monosaccharides. The selected mutants showed a significant reduction in the cell wall material, particularly in the more easily extractable fractions, the most affected polymers being potentially pectin-like structures. As pectin has a significant effect on cell wall permeability, reduced quantities of this polymer have possibly led to increased permeability of the dye during the mutant selection. Nevertheless, increased permeability to small molecules does not necessarily correlate with increased accessibility to larger molecules, as required for genetic transformation, or a weaker cell wall, more easily disrupted during metabolite extraction. Further research is required to investigate the impact of the observed changes in cell wall composition on the efficiency of transformation and of product extraction of the mutants. On the other hand, these lines might represent a useful tool to investigate the genetic basis of C. vulgaris cell wall biosynthesis, which might be used to identify targets for strain improvement.