Quest for the key to malic acid production by Aspergillus oryzae

Abstract

The petrochemical industry has been the focus of research and development for many years and is the norm for many chemical processes. The potential to produce bulk valuable chemical precursors, like malic acid, from microbial bioconversion has been proposed for many years but is still being researched to a lesser extent. Malic acid is a speciality chemical that is mainly used in the food and beverage industry. Aspergillus oryzae has shown to be a natural producer of malic acid using various renewable sugar substrates and nitrogen sources. Before venturing into this avenue for biomass-based production of malic acid, the influence of process parameters on scalability needs to be investigated. The dissertation aimed to: (i) investigate the trigger for malic acid production by A. Oryzae; and (ii) classify the driving force for this. This was tested in either a novel bio-reactor that used immobilised A. Oryzae NRRL 3488 or shake flasks experiments. The fermentation strategy tested various morphologies (fungal and buffer), 1-step vs 2-step acid cultures, and various types of buffers such as potassium phosphate, NaOH, CaCO3, and MgCO3. The influence of different CaCO3 concentrations was also investigated. A two-phase fermentation strategy in the bio-reactor using NaOH as neutralising agent resulted in malic acid production. However, after 205 hours only 6 % of the initial glucose was consumed with 3.34 g/L of malic acid produced which was significantly lower than the amounts reported in the literature. Citric acid was the main catabolite produced followed by malic acid and then pyruvic acid. To investigate the influence of morphology and pH control on malic acid production, shake flasks were employed that used pellet or immobilised morphology with a buffer. Fermentations with a phosphate buffer and pellet morphology didn’t produce any malic acid or by-products and were ineffective at maintaining the pH above 6 (after 48 hours pH was below 5). The addition of 40 g/L NaHCO3 with the phosphate buffer improved pH control for both immobilised and pellet morphology experiments and resulted in the production of organic acids. The 2 different morphologies yielded similar results for organic acid production with malic acid the main product followed by succinic acid, citric acid, and then ethanol which was not previously reported as a by-product. The one-step fermentation method was validated in shake flasks with dosing of CaCO3 whereby it was seen that flasks with higher concentrations of CaCO3 measured higher concentrations of malic acid. The influence of CaCO3 concentration on the ability of A. oryzae to produce malic acid was investigated using 3 different concentrations: 20 g/L, 80 g/L, and 120 g/L CaCO3. It was found that 120 g/L CaCO3 performed the best with 89 % of the glucose consumed where 45 % was converted to malic acid. Following was 80 g/L CaCO3 with 78 % glucose consumption (35 % to malic acid) and lastly 20 g/L CaCO3 with 68 % of the glucose consumed (19 % to malic acid). It was also found that up to 4 times more biomass was produced with 80 g/L and 120 g/L CaCO3 when compared to 20 g/L. Of interest, it was decided to add powdered CaCO3 at the start of the fermentation, after 24 h and in pellet form. It was found that this did not significantly improve the amount of malic acid produced but did change the morphology of the biomass (filamentous instead of pellet). These results suggested that CaCO3 played a more significant role than just controlling the pH. Further experiments showed similar results when MgCO3 was used in the same carbonate ratio as CaCO3. MgCO3 had a lower glucose consumption rate (50 % after 300 hours) when compared to CaCO3 where all of the glucose was consumed after 200 hours. Samples measured before and after acidification with HCl showed no significant difference in the amount of malic acid measured which suggested the calcium-malate precipitation mechanism was not an influencing factor as previously hypothesised. The study tested various parameters to find the key to malic acid production with A. Oryzae and found that the role of CaCO3 was an important part of the map.