Understanding the effect of key operational conditions on amino acid acidification for a knowledge-driven protein fermentation

Resumo

The depletion of fossil fuels and the growing need for a more sustainable, circular economy is paving the way to the concepts of biorefinery and resource recovery. In the biorefinery framework, the anaerobic digestion for the production of biogas and biomethane is an already established technology. However, the application of those products is generally limited to energy generation and the associated profitability is quite low. The carboxylate platform approach poses an interesting alternative, allowing to convert organic wastes to volatile fatty acids, which are then further refined to a variety of end-products (pharmaceuticals, bioplastics, etc). Volatile fatty acids are produced in mixed-culture fermentation processes, whose application has been thoroughly studied for sugar and carbohydrates-rich substrates. Conversely, the knowledge concerning the anaerobic fermentation of proteins is limited and sometimes contradictory. In particular, little information is available on how the operational conditions specifically affect the consumption and transformation of the different amino acids and their interactions. Therefore, the goal of this PhD thesis is to understand the effect of key operational conditions on the conversion of amino acids to volatile fatty acids. The focus is placed on the effect of substrate composition, operational pH and micronutrients supplementation on the process. Beyond the fermentation of proteins, the opportunities of using carbohydrates as a cosubstrate are explored together with the feasibility of chain elongation processes leading to longer chain fatty acids production. The results obtained highlight protein composition as one factor determining both the overall conversion of the amino acids to volatile fatty acids and the resulting products spectra. In fact, preferential consumption was identified during casein and gelatin fermentation, probably as a result of amino acids interactions. Their redox roles appear to be influential in determining the outcome of the process, as a surplus in electron donor amino acids seems to promote the overall acidification. pH was identified as a key parameter as well. Neutral conditions are the most favourable for the microbial community, as protein conversion is maximised regardless of the amino acid profile. High pH values are associated with increased acetic acid formation, whereas acid conditions lead to a more diverse product spectrum. In particular, the production of longer chain volatile fatty acids is promoted at low pH, partially as a result of chain elongation processes. Interestingly, the effect of pH depends on the protein composition, as casein fermentation was influenced more strongly than in gelatin case. The supplementation of micronutrients promotes the overall conversion of amino acids to volatile fatty acids only at neutral pH, favouring the occurrence of secondary processes such as chain elongation reactions and the isomerisation between linear and branched forms of the carboxylic acids. The supplementation of sugars is another viable strategy to steer the selectivity of protein fermentation towards the production of longer chain volatile fatty acids, such as n-butyric and especially n-valeric acid. Moreover, protein conversion is not affected for sugar-to-protein ratios equal or lower than 1. Conversely, higher sugar loadings appear to hinder amino acids consumption while favouring the production of short chain volatile fatty acids and secondary metabolites (e.g. ethanol). Interestingly, these changes are reversible given that lowering the sugar-to-protein ratio allows to progressively return to the previous products distribution. Chain elongation processes based on amino acid consumption were also detected during casein monofermentation at acid conditions, and the metabolic pathways associated with these processes were conceptualised and described. It is hypothesised that chain elongation reactions were used by the microbial community as a way to reduce the toxicity exerted by the acid equivalents at low pH by taking advantage of the excess of reducing power generated by casein conversion. Micronutrients supplementation enables chain elongation occurrence for different proteins (i.e. gelatin) and different pH values (i.e. 7), while the cofermentation of proteins with sugars further promotes the process by providing a surplus of electron donor compounds (i.e. ethanol and lactate) and reducing power. In conclusion, the knowledge acquired in this thesis is useful for the design and optimisation of mixed-culture fermentation processes using protein-rich streams, as it will help to define the operational strategies to steer their fermentation towards the desired outcome.