thesis

Single cell oil production: a new approach in biorefinery

Abstract

A socially responsible economic growth, devoted to the future generations, requires long-term secure and available resources for industrial production, in terms of raw materials, energy and water. It should be environmentally friendly and a forward-looking financial system capable of future challenges with a global point of view. However, the present economic model based on the non-renewable fossil resources (oil, natural gas, coal, minerals) for energy and industrial production is the reason of energy instability, climate changes and therefore it cannot be considered sustainable. In this context, biotechnological techniques, such as the biorefinery are becoming more attractive. The biorefinery consist in the sustainable transformation of biomass, such as plant, algae, yeast, bacteria, into a wide range of marketable products and, in the mean time, into energy. Oils derived from biomass sources are named Microbial Oils, Unicellular Oils or Single Cell Oils (SCOs). The sustainable production of Single Cell Oils (SCOs) has garnered recent attention. The goal is new strategies or the biochemical/microbial conversion processes in order to increase their productivity and competitiveness. The identified strategies include the metabolic and genetic engineering of microorganisms, new fermentation technologies, innovative process choices, low-value feedstock and the recycle of by-products. SCOs are produced by oleaginous microorganisms which are able to accumulate between 20% and up to 80% lipid per dry biomass in the stationary growth phase under nutrient limitations, e.g., nitrogen or phosphorus, with simultaneous excess of carbon source. Depending on the oleaginous microorganisms including bacteria, yeasts, microalgae or fungal species, fatty acid profile of SCOs can vary making them highly suitable for different industrial applications. The SCOs, obtained from plant and microbial sources, offer several advantages, including faster production, less labor, more season and climate flexibility, and easier scale-up. The aim of this thesis is to explore the capacity, efficiency and productivity of oleaginous microorganisms, grown on agro-industrial lignocellulosic biomasses, to accumulate Single Cell Oils. This allow to reduce the environmental problem associated with the recycle of residues of various industrial processes allowing while increase the economic advantage linked to the SCO production. In order to make Single Cell Oil production more economical and sustainable, the experimental activities were aimed to achieve the following objectives: - The optimization of operating conditions of enzymatic hydrolysis evaluating the effects of enzyme concentration, temperature and pH on the fermentable sugar production. The experimental tests were carried out to assess the viability of innovative fermentation processes, i.e. in single stage or single reactor, with the aim to reduce the capital costs. It was found the synergism of enzymatic mix as well as the positive effects related to a lower temperature, suspended composition of hydrolysates, pH control and reaction time. - The implementation of enzymatic hydrolysis and oleaginous fermentation in single reactor (SRF) that offers an useful option to integrate in a single reactor two different stages of the microbial oil production: the enzymatic hydrolysis of the pretreated lignocellulosic biomass and the microbial fermentation of the obtained fermentable sugar mixture. Specific glucose consumption rate (μs), lipid yield (Ylipid) were calculated. The results suggest positive potential application of such process that still remains unexplored for Single Cell Oil production but demonstrate that they are suitable for biodiesel, bioplastic production or for other products of industrial interest. - The investigation of synergistic effect of yeast-microalga mixed cultures, in order to find suitable operating conditions to improve SCO production in mixotrophic microbial cultures. To test the consortium in clearly defined conditions, a synthetic medium was developed that integrates necessary elements of known culture media for both organisms, with the use of pH control. Growth series were done in batch, under constant light, agitation and temperature, and monitored gas exchange. In the model system, symbiotic growth was observed of the consortium with synergistic effects on biomass yield. Similar results were obtained in a system using lignocellulosic hydrolysate, except for an increase in lag phase due the presence of inhibitors. Growth even in anaerobic conditions (N2) confirmed synergistic interactions between the microalga and the oleaginous yeast. The performance of the consortium under different conditions is discussed in terms of growth rate, biomass production and lipid content of the biomass. - The implementation of yeast-microalga mixed cultures in open pond, overcoming contamination problems. The yeast grew, reaching the maximum concentration just after few days. The rapid growth kinetic was expected for the yeast while the very low cell proliferation can be explained by the low quantity of organic carbon in the medium

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