Loffredo, Raffaele (2026) Succinic acid production from organic waste. [Tesi di dottorato]

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Abstract

Succinic acid is a dicarboxylic acid widely used as a building block in several industrial sectors, including pharmaceuticals, cosmetics, and the chemical industry. It is a well-known precursor of polybutylene succinate, a biodegradable polymer employed in the production of bioplastics. Since the early 2000s, succinic acid has been recognized as a high-value platform chemical and was included in the Top Value-Added Chemicals list by the United States Department of Energy (DOE), owing to its potential for production from renewable biomass. Currently, most of succinic acid is produced from non-renewable petrochemical sources, such as maleic acid or maleic anhydride, which are derived from fossil resources. This conventional production route is increasingly unsustainable due to several factors, including the volatility of fossil fuel prices and the significant environmental impact associated with their use, particularly greenhouse gas emissions, mainly CO2. To address these limitations, alternative biological production routes have been extensively investigated. Succinic acid can be produced as a metabolic intermediate in animal and plant tissues and by various microorganisms. Among these, bacteria and yeasts are of particular interest due to their ability to naturally produce high concentrations of succinic acid and their suitability for industrial fermentation processes. Actinobacillus succinogenes is a facultative anaerobic, capnophilic, non-pathogenic, Gram-negative, and non-motile bacterium classified as a biosafety level 1 (BSL-1) microorganism. Originally isolated from the bovine rumen and belonging to the Pasteurellaceae family, this strain exhibits remarkable metabolic versatility, being able to utilize a wide range of carbon sources, including glucose, xylose, arabinose, mannose, galactose, fructose, sucrose, lactose, cellobiose, mannitol, maltose, and glycerol. Moreover, A. succinogenes is capable of naturally producing succinic acid at high concentrations, making it a highly promising candidate for industrial-scale bio-succinic acid production. This capability of the strain to metabolize a wide range sugars enables the use of lignocellulosic biomass as a renewable feedstock. Lignocellulosic biomass is abundantly available, as it largely consists of residues from agro-industrial processes, and its valorization aligns with circular economy principles. However, fermentable sugars such as glucose and xylose are not readily available in lignocellulosic biomass, as they are embedded within a complex and recalcitrant matrix composed primarily of cellulose, hemicellulose, and lignin. Consequently, pretreatment processes are required to disrupt this structure and release fermentable sugars. These pretreatments often involve harsh conditions, including strong acidic or alkaline environments, high temperatures, and high pressures, which can lead to the formation of inhibitory compounds such as acetic acid and 5-hydroxymethylfurfural. These inhibitors negatively affect microbial growth and fermentation performance. In addition, during the fermentation process for succinic acid production, several by-products are generated, with acetic, formic, and lactic acids being the most common. The presence of these compounds in the fermentation broth complicates downstream processing, making the separation and purification of succinic acid technically challenging and economically demanding. This thesis investigates the feasibility of producing succinic acid via fermentation from lignocellulosic biomass, specifically wheat straw, as the primary feedstock. In the first phase of the study, different initial sugar concentrations and microbial strains were evaluated in a synthetic medium to identify the optimal balance between substrate availability and microbial performance. Subsequently, the growth and fermentative capabilities of Actinobacillus succinogenes were assessed in a synthetic fermentation medium supplemented with inhibitory compounds to simulate the conditions typically found in hydrolysates obtained from the steam explosion of wheat straw. In the initial experiments, no additional carbon or nitrogen sources were supplied. Under these conditions, the strain exhibited strong inhibition, prompting the investigation of yeast extract and magnesium carbonate supplementation as strategies to enhance succinic acid production performance. In the final fermentation trials, different dilutions of wheat straw hydrolysate were tested, and the concentrations of yeast extract and magnesium carbonate were optimized to avoid unnecessary supplementation while maintaining beneficial effects on fermentation efficiency. Finally, an innovative approach for reducing by-product accumulation was explored. This strategy involved a second-stage fermentation process employing Cupriavidus necator, a bacterium capable of utilizing acetic and formic acids as carbon sources to produce polyhydroxybutyrate (PHB), another high-value bioproduct. This integrated bioprocess aims to decrease the concentration of inhibitory by-products, limit the reliance on costly downstream purification techniques, and improve the overall economic feasibility of bio-based succinic acid production through the co-production of an additional marketable compound.

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