Innovative biotechnological process for the conversion of lignocellulosic biomasses into high added-value products.

Climate change and environment degradation are an concrete threats for the world. To overcome these threats, the European Union (EU) has put in place the European Green Deal (EGD), a growth strategy aimed at developing an EU competitive and sustainable economy. The EGD, adopted at the end of 2019, provides the action plan to achieve the carbon neutrality, resource efficiency and zero pollution in the EU by 2050. This set of policy initiatives, coupled with the new EU Recovery Plan developed to support the repair of the economic and social damages caused by the COVID-19 pandemic, represents today the most crucial milestones in the green transition towards a more resilient, competitive and environmentally sustainable Europe. The transition from a fossil-based economy to a bio-based economy is necessary to guarantee a sustainable future for the society. In this sense, the development of biorefinery processes able to replace traditional fossil processes has attracted great attention. Biorefineries are technological platforms making use of both biotechnological and/or chemical processes to convert different bioresources into a spectrum of high value-added products. One of the most widespread and consolidated biomass biorefinery processes concerns the production of first-generation biofuels from vegetable oils, but the environmental externalities due to the intensive cultivation of oil crops has led several European countries to discourage the use of some vegetable oils for biofuels production. An excellent, green, and sustainable alternative to vegetable oils is represented by microbial oils produced by oleaginous microorganisms. Some microorganisms are defined oleaginous in consequence of their ability to accumulate lipids for over 20% of their dry cellular weight. This group includes several eukaryotic microorganisms (such as fungi, yeasts and algae) and some species of autotrophic and heterotrophic bacteria able to accumulate lipids in the form of triglyceride (TAG). Among these, the yeasts are considered the best candidates for industrial processes thanks to their rapid growth, the ability to utilize a wide variety of raw materials and the easy cultivation in large fermenters. The lipids produced by oleaginous yeasts, better known as Single Cell Oil (SCOs), have a composition very similar to vegetable oils, with a predominance of oleic acid (18:1), followed by palmitic acid (C16:0), linoleic acid (C18:2), and stearic acid (C18:0). Of the 1500 known species belonging to 100 different genera, about 30 of them are capable of accumulating lipids, and the most known oleaginous yeasts include the genera Yarrowia, Rhodotorula (Rhodosporidium), Lipomyces, and Cryptococcus. SCOs may serve as a renewable source of edible oil and as an intermediate ‘‘building block’’ for oleochemicals, including: fuels, soaps, plastics, paints, detergents, textiles, rubber, surfactants, lubricants, additives for the food and cosmetic industry and many other chemicals. Although the use of SCOs as a feedstock for biofuels and biochemicals has received interest in recent years, high production costs, estimated in the range 4.1 and 5.8 $ / kg, prevented their industrial applications. Using low-cost carbon sources together with more efficient bioconversion processes could make this process more competitive. Several types of waste and low-cost carbon sources, including olive mill wastewater, and by-products of the dairy industry, food processing, and lignocellulosic residues have been used as feedstock to produce microbial lipids. Due to the high content of inorganic material, widely available lignocellulosic residues such as agricultural straws could be more suitable to achieve fermentable sugars with respect to produce heat and electricity through thermal processes. Due to their recalcitrant nature, lignocellulosic biomasses require pre-treatments to favour the breakdown of the lignin structural matrix and favour the enzymatic hydrolysis of polysaccharides. Pre-treatment involves the application of physical, chemical, biological or physicochemical approaches, among which steam explosion has been recognized as one of the most effective, economical and sustainable processes. The main limit of this technology is the formation of thermal degradation by-products, such as 5-hydroxymethylfurfural (5-HMF), furfural, weak acids, and phenols. These molecules could inhibit the growth of most microorganisms. For these reasons scientific efforts are aimed at identifying robust microorganisms able of tolerating the stress induced by these molecules. In addition, genetic engineering, adaptive evolutionary techniques, and fermentation strategies could improve the specific microorganism resistance and increase the process yields. This PhD was supported by regional government in the framework of the industry 4.0 strategy and, in order to meet the needs of the Basilicata region, the main objective of this PhD activity was the development of a process which might be useful for regional economy. By considering the strong agricultural vocation of Basilicata region and the presence in this area of one of the largest oil fields in Europe, this PhD project was aimed to develop a biorefinery process for valorisation of agricultural wastes, for the production of high added-value products. The development of a biorefinery process useful for enhancing the local agriculture by-products represents a green and sustainable alternative to classic petrochemical processes. The aim of this Ph.D. thesis was the investigation and the optimization of innovative processes to convert lignocellulosic residues into microbial oils at industrial relevant conditions. In fact the profitable conversion of lignocellulosic biomass into biobased products through the platform sugars requires the intermediate production of concentrated biomass hydrolysates that, however, could contain many microbial inhibitors. The development of optimized bioconversions processes could alleviate the production costs for the hydrolysates detoxification. In particular, the research work concerned the conversion of second-generation sugars through previously optimized processes of steam pretreatment and enzymatic hydrolysis of wheat straw and cardoon residues into microbial oils. Teste microorganisms included oleaginous yeasts belonging to the species Cutaneotrichosporon curvatus CA-3802, Lipomyces tetrasporus Li-0407, and Yarrowia lipolytica ATCC 46483. Many fermentation set-up were developed to overcome the inhibition induced by thermal degradation by-products and maximize the conversion yields. The fermentation process optimized in shaken flasks was then scaled-up in the 2, 10 and 50 L stirred tank bioreactor. According to sustainable chemistry strategies and to improve the economic feasibility of the process, the downstream phase was implemented through the use of sustainable and non-hazardous solvents. Furthermore, the suitability of the microbial oil versus target applications was investigated for the production of biodiesel, green diesel and polyurethanes. Finally, on the basis of the biorefinery process developed in this Ph.D. thesis, a technical-economic analysis was carried out. Specific aims of the research project were the following: I. To study and optimize the growth of oleaginous yeasts in simulated medium and un-detoxified biomass hydrolysates, and improvement of the microbial conversion in order to produce high-titer lipids; II. scale-up of the biorefinery process from laboratory to pilot scale; III. evaluate an innovative extraction mixture and synthesize high added-value products, such as intermediates for the food and oleochemical industries; IV. to evaluate the overall economic feasibility of the implemented process through a technical-economic analysis; In particular, Chapter I describes the state of the art of the proposed process with particular interest on the use of oleaginous yeasts for the production of SCOs. Chapters II and III describe the microbial conversion of undetoxified lignocellulosic hydrolysates, such as cardoon residues hydrolysate and wheat straw hydrolysate, by the use of Cutaneotrichosporon curvatus CA-3802, Lipomyces tetrasporus Li-0407, and Yarrowia lipolytica ATCC 46483. The activities were focused on the process conditions useful to overcome the inhibition induced by the biomass degradation products. Furthermore, the microbial lipids obtained were characterized and converted into high added-value products. Chapter IV describe the implementation of economic feasibility and scale-up of the process. The first step was addressed to the optimization of enzymatic hydrolysis process of steam pretreated wheat straw in order to enhance economic feasibility of the process through the decrease of the enzymatic load. Subsequently, cheap nitrogen sources were also evaluated in the lipid synthesis phase. The scale-up of the process was carried out in pilot scale 50 L bioreactor. Lipids extraction and synthesis of high added-value products were described in Chapter V. An innovative extraction solvent was studied. Microbial lipids produced were converted into advanced fuels for hydrocarbon industry and microbial bioplastics. Finally, the technical-economic analysis of the proposed biorefinery process was described in Chapter VI.