Abstract
Pyrolytic aqueous condensate (PAC) might serve as a cost-effective substrate for microbial malic acid production, as it is an unused side stream of the fast pyrolysis of lignocellulosic biomass that contains acetol and acetate as potential carbon sources. In the present study, shake flask cultures were performed to evaluate the suitability of acetol and its combination with acetate as substrates for growth and L-malate production with the filamentous fungus Aspergillus oryzae. Acetol concentrations of up to 40 g/L were shown to be utilized for fungal growth. In combination with acetate, co-metabolization of both substrates for biomass and malate formation was observed, although the maximum tolerated acetol concentration decreased to 20 g/L. Furthermore, malate production on PAC detoxified by a combination of rotary evaporation, overliming and activated carbon treatment was studied. In shake flasks, cultivation using 100% PAC resulted in the production of 3.37 ± 0.61 g/L malate, which was considerably improved by pH adjustment up to 9.77 ± 0.55 g/L. A successful scale-up to 0.5-L bioreactors was conducted, achieving comparable yields and productivities to the shake flask cultures. Accordingly, fungal malate production using PAC was successfully demonstrated, paving the way for a bio-based production of the acid.
Highlights
Since malic acid is an intermediate of the tricarboxylic acid (TCA) cycle and an essential component of the metabolism in aerobic cells, many microorganisms are naturally capable of producing this acid, or can be enabled to do so by metabolic engineering [16]
The introduction and heterologous expression of genes from other organisms has been carried out to enhance fungal acid production [71,72] and even offers the possibility of completely reconstructing metabolic pathways. Such an approach could potentially be pursued to allow malate production on acetol, which would both eliminate the need to remove the ketone by pretreatment and allow more carbon in the condensate to be used for malate formation. It was shown for the first time that A. oryzae is able to use acetol as substrate for growth in shake flask cultures
The maximum tolerated concentration was identified to be 40 g/L, increasing inhibition was already observed for lower acetol concentrations
Summary
Malic acid is a dicarboxylic acid used primarily as an acidulant and flavor enhancer in the food and beverage industry, but it can be applied as an ingredient in cosmetics and pharmaceuticals [1–4], as well as in animal feed [5–7]. Another potential field of application, which could become increasingly important in the future, is its use in the production of biopolymers [8,9]. Since malic acid is an intermediate of the tricarboxylic acid (TCA) cycle and an essential component of the metabolism in aerobic cells, many microorganisms are naturally capable of producing this acid, or can be enabled to do so by metabolic engineering [16]. Identifying cost-effective alternatives to the Received: 17 February 2022
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