Integration of fermentation and cooling crystallisation to produce organic acids

Abstract

Fermentation products are gaining more attention in the last years due to the fact that the metabolic and genetic engineering field has been developing techniques to enhance fermentation yields and make biochemical processes competitive compared to traditional chemical production. However, as fermentation productivities are becoming higher, many fermentation processes suffer from product inhibition and/or toxicity. Moreover, besides the inhibition/toxicity problem, the complexity of the fermentation media makes the process recovery an important step in the development of the whole production process. In this thesis, product recovery applying crystallisation techniques was studied for two organic acids: fumaric acid and 7 aminodeacetoxycephalosporanic acid (7-ADCA). In the case of fumaric acid one of the main problems of the conventional fermentation is that it is carried out at a pH in the range of 4.5–6.5, leading to fumarate salts instead of undissociated fumaric acid. Stoichiometric amounts of bases such as NaOH and CaCO3 are added during fermentation to control the pH. To recover fumaric acid, the supernatant has to be acidified, for example with stoichiometric amounts of sulphuric acid, so that undissociated fumaric acid can precipitate. Stoichiometric amounts of waste salts such as sodium or calcium sulphate are formed in this manner. A similar problem is present in the 7-ADCA production. Here the fermentation and deacylation are working in a pH range of 6.0 – 8.0 while the crystallisation recovery needs to be at pH 4.0. This implies the addition of mineral acids to decrease the pH, and hence waste salts are produced. To overcome these problems, we integrated fermentation and cooling crystallisation to recover the product. At lower temperature the fermentation product should become partly insoluble, so that it can be filtered off, thus avoiding acidification and waste salts production. In this thesis Chapter 1 presents general aspects of the In-Situ Product Recovery (ISPR) techniques and their development as process integration tools. Additionally, a general overview of the fumaric acid and 7-ADCA systems is presented to show the concept that is developed in this research. Chapters 2-5 deal with the fumaric acid system and chapter 6 with the 7-ACDA system. Chapter 2 reviews the production of fumaric acid by fermentation. Here the metabolism of Rhizopus oryzae (the strain used in this study for fumaric acid production) and the importance of CO2 fixation in this mechanism are described. Future options for improvement in the fermentation and recovery are also discussed here. Fermentation development for low pH values is presented in Chapter 3. This chapter shows that delay of pH control during the fermentation until a low pH has been reached at the end of the batch cycle does not affect fermentation productivities and allows the fermentation unit to be integrated with product crystallisation without pH shifting. Nonetheless, because it is very important to understand the conditions leading to crystallisation of fumaric acid or its sodium salts, the solubility behaviour of fumaric acids and its sodium salts are studied and presented in Chapter 4. The findings here showed that the integration should be performed in a pH range of 2.5 – 3.5 in order to get only fumaric acid crystals and avoid the crystallisation of one of the sodium fumarate salts. Integration of fumaric acid production by fermentation and cooling crystallisation is proven to work in an experimental set-up that is described in Chapter 5. In this chapter it is also shown that the integration of these two units can be done while avoiding pH shifting. Consumption of neutralising agent is reduced as compared to the base case system, which uses sulphuric acid to precipitate the fumaric acid out of the fermentation broth thus reducing the associated production of waste salts. A perspective of industrial scale-up of the experimental set up is presented, showing that the studied process may become competitive at industrial levels if the some elements in the concept (like lower pH) are improved further. In Chapter 6 the production of 7-ADCA is investigated. A new integrated process option for ?-lactam nuclei production is proposed, which is an integration of fermentative production and enzymatic deacylation of adipyl-7-aminodeacetoxycephalosporanic acid (adADCA) in one reactor, thus producing 7 ADCA directly from glucose. The outcome of this study proves that according to mathematical modelling it is possible to recover 7-ADCA via cooling crystallisation and in this way the number of downstream steps can be reduced. This new process option avoids the use of mineral acids and bases for pH shifts and leads to a reduction in waste salt production. In Chapter 7 the outlook of this thesis and recommendations for future work are presented.