WINERY WASTEWATER TREATMENT BY MICROALGAE CO-CULTURE FOR LOW-COST BIOMASS PRODUCTION IN A BIOREFINERY CONCEPT

The growing industrialization, the high level of pollution, and the reduction of resources lead to the necessity to find an alternative way to handle the entire production chain from the raw materials to the finished products. The zero-waste strategy and the circular economy are the best solutions to remediate the dramatic conditions in which our planet pours. Moreover, the reduction of fossil fuels reserves and the continuously increasing demand for energy around the world has led to the necessity to find an eco-sustainable alternative to conventional fuels. In the last few years, biofuel production from different plant sources has been increasingly studied by researchers. The production of third-generation biofuels from raw materials that do not compete with food crops is attracting more and more attention. Third-generation biofuels can be produced from microalgal biomasses or from their intracellular components such as lipids. Moreover, their production, if compared to conventional biomasses, reduces land and water utilization along with the use of pesticides. Microalgae are unicellular microorganisms able to grow under autotrophic, heterotrophic or mixotrophic conditions depending on the carbon source used in their metabolism as well as light conditions. They are composed mainly of lipids, proteins and carbohydrates, whose relative proportions depend in particular on the species and growth conditions. They are generally used for human or animal nutrition, or extraction of added-values components for chemical and pharmaceutical industries, but also for biofuel production. In recent years, lipid, protein, and pigment extraction from microalgae has been widely studied for various applications such as the productions of biodiesel from the lipid fraction and of nutraceuticals and dyes from vitamins, proteins, and pigments. However, it is important to stress that the microalgae biorefinery concept is the only way to make microalgae competitive with products obtained from conventional sources, and that the use of microalgae to produce only biofuels or only nutraceuticals has not yet reached clear-cut economic feasibility. The biggest challenges are the relatively high cost of biomass production and the energy demand for the extraction and separation processes. Therefore, in order to make microalgae products economically viable and increase their marketability, it is necessary to reduce costs, for example by valorizing process residues as co-products. It is generally accepted that the sale of co-products will make the production of biofuels from microalgae economically feasible. Indeed, it was estimated that the residue of microalgal biomass after lipid extraction could be worth between 100 and 225 USD per ton and could yield co-products ranging in value from 0.95 to 2.43 USD per gallon of biodiesel produced. Moreover, the microalgae protein fraction has an economic value that ranges from 0.86 USD/kg as feed to 5.57 USD/kg as food. Moreover, thanks to their capability of also metabolizing organic carbon, microalgae can in fact be grown in wastewaters, thus reducing the use of fresh water, the cost of growth medium, the energy consumption and, at the same time, the wastewater polluting impact. There are several studies in the literature focusing on the use of microalgae to treat wastewaters such as municipal and textile wastewaters, among others. In this contest, agri-food wastewaters are good candidates to be used as microalgae medium because they are rich in nutrients and the resulting biomass obtained after treatment could be used for the extraction of high added value components, such as protein and pigments. Between them, winery wastewaters (WWWs) which are released from different activities of the wine making process, namely tank washing, transfer, bottling and filtration, are suitable to be treated by microalgae. The polluting impact of WWWs is related to their high organic load (polyphenolic compounds, sugars, organic acids and esters), low pH (3–5), high content of suspended particles and large volumes (0.5–14 L per liter of wine produced). Owing to the release of organic compounds and inorganic ions, their disposal in land without adequate treatment can change the physicochemical properties of groundwater such as color, pH and electrical conductivity, among others. Whit regard to the open issues recalled above, the research project has aimed to develop a biorefinery from microalgae. Winery wastewaters were used as growth medium both for microalgal biomass production and reduction of pollutant impact, the protein fraction was extracted from the biomass as high added-value component, and the residual biomass was submitted to pyrolysis process to produce biofuels. The topic addressed by this thesis is organized and subdivided into chapters as follows. Chapter 1- Literature review on the biorefinery concepts, the application to microalgae production and the microalgae world situation concerning their metabolisms, growth system, industrial application (wastewaters treatment, extraction of high-added-value components and, biofuels production). Chapter 2- The optimization of winery wastewaters concentration in microalgae growth medium to obtain both high microalgae concentration and productivity and good results in terms of reduction of pollutant impact. Three different winery wastewaters collected from different steps of the winemaking process were studied. The co-culture of Chlorella vulgaris and Arthrospira platensis was grown under continuous light and air supply. The optimized parameters obtained in the previous section were studied at different light conditions to identify the prevalent metabolism of the microalgae to consume the pollutant molecules present in the wastewaters. Moreover, the microalgae growth was performed into different photobioreactor configurations: tubular photobioreactor (TP), column photobioreactor (CP) and, open pond (OP) to improve the biomass concentration and the pollutant impact removal efficiency. Chapter 3- To perform a scale-up of the process the co-culture was grown in 20 L column photobioreactor, the growth medium under the condition optimized in the previous chapters was supplied continuously by a pump system. Chapter 4- The extraction of high-added value components from microalgal biomass were investigated taking into account the biorefinery concept. The optimization of the protein extraction process from A. platensis by Ultrasound-Assisted Extraction was performed using Box-Behnken Design in which the effects of extraction time, solvent volume, and mass of A. platensis were investigated. Moreover, the extraction and purification of c-phycocyanin from A. platensis and the subsequent protein extraction on wet c-phycocyanin residue was performed. The protein extraction from the co-culture grown in the different photobioreactor configurations was carried out under the condition optimized in the previous chapter. Moreover, the effect on cell size and cell wall thickness of growing the co-culture in presence of winery wastewaters was investigated. Variation of protein expression as function of photobioreactor configuration was also assessed. Chapter 5- The energetical recovery of the produced co-culture biomass was investigated. The pyrolysis process was carried out on microalgal biomass obtained from wastewater treatment in membrane photobioreactor. The operational condition of the process and, the distribution of reaction products and their composition ware studied. Chapter 6- A final discussion of the process was performed based on the results obtained from the previous chapters.