Novel membrane-based biotechnological alternative process for succinic acid production and chemical synthesis of bio-based poly (butylene succinate)
Introduction
Succinic acid (1,4-butanedioic acid) is important for the synthesis of some valuable chemical derivatives, which can be used in food and pharmaceutical products, solvents, biodegradable polymers, surfactants and detergents (Zeikus et al., 1999, Bozell and Petersen, 2010). Due to its independence from petroleum, environmental benefit and CO2 sequestration, biological production of succinic acid has been investigated intensively during recent years (Song and Lee, 2006, Cheng et al., 2012b).
Great efforts have been devoted to develop fermentation technology to produce succinic acid as replacement of the petrochemical route (Guettler et al., 1999). Efforts mainly focus on three aspects: development of efficient biocatalysts with higher succinic acid productivity (Kim et al., 2008, Liang et al., 2013), fermentation control (Li et al., 2010b) and downstream process improvement (Cheng et al., 2012a) aimed to separate succinic acid effectively from fermentation broth. Among succinic acid producing bacteria, Actinobacillus succinogenes and genetically engineered Escherichia coli have been mostly studied and showed great advantages in succinic acid production (Guettler et al., 1999, Millard et al., 1996). However, product inhibition was observed both in A. succinogenes and E. coli because of the acids accumulation and nutrient depletion, which limits the bacterial growth rate and productivity. For A. succinogenes, cell growth was inhibited at concentration levels of 8.8–17.6 g/L formate, 10–40 g/L acetate, 9–18 g/L lactate, and 10–80 g/L succinate (Li et al., 2010a). E. coli is more resistant to acidic products than A. succinogenes but still suffers from products inhibition. In this situation, novel bioreactors and integrated fermentation and separation processes which can remove produced acids and replenish new fermentation media should be developed.
The downstream process consists of preliminary removal of biomass and larger molecules, decolourization, conversion of succinate salts into free acid, and crystallization. The removal of biomass and lager molecules such as proteins is crucial to the following purification step. In recent years, ultrafiltration has been widely used in various physicochemical and biochemical processes for the separation of solids from liquid at low operational cost, low energy consumption and with elimination of filter aids. It has been demonstrated that clarification of succinic acid fermentation broth by ultrafiltration is possible (Wang et al., 2012b). Compared to the traditional centrifugation, clearer fermentation broth can be achieved by ultrafiltration. Our previous study indicated that 100% cells and 90% protein can be getting out fermentation broth while for centrifugation only 92% cell and 53% protein can be removed (Wang et al., 2012a). More importantly, membrane systems can be connected to the fermentation bioreactor to alleviate product inhibition and achieve high cell density. Permeation was drawn off continuously and then fresh media was added by the coupled system. It is reported that the submerged membrane fermentation could double lactic acid production and cell density (Ramchandran et al., 2012). An integrated membrane-bioreactor-electrodialysis system was also built for succinic acid production at high concentration, productivity and yield. Under the optimized conditions, biomass concentration and succinate concentration reached 42 g/L and 14.8 g/L, which are respectively 28 and 20 times higher compared to batch cultures (Meynial-Salles et al., 2008). In this study, ultrafiltration membrane was applied to connect fermentation and separation process, and to clarify the fermentation broth.
The key challenges in the separation process are the low concentration of succinic acid in the aqueous broth, the presence of the byproducts such as lactic acid, acetic acid and formic acid with physicochemical properties similar to succinic acid. Up to now, several recovery technologies such as adsorption, extraction and electrolysis have been investigated for the recovery of succinic acid (Pratiwi et al., 2013, Orjuela et al., 2013). Crystallization has been proved to be effective in getting succinic acid from the aqueous broth even in the presence of formic acid and acetic acid. However, direct crystallization by acidification was proven to yield low recoveries and purities (Lin et al., 2012). Resin-based crystallisation could improve the yield and purity for the separation process (Lin et al., 2010). In order to get succinic acid of higher purity, fermentation broth should be treated before crystallization. To develop a cost-effective downstream process, not only a single operation unit but also the compatibility of these operation units especially the connection possibility of these units should be considered.
Succinic acid can be transformed into a wide range of chemicals and polymers (Delhomme et al., 2009). Among them, PBS has attracted much attention from both academia and industry because of its biodegradability, excellent thermal ability and good mechanical properties (Sinha Ray et al., 2003) PBS exhibits ecological advantages over non-biodegradable polymeric material. Currently, commercially available PBS is efficiently synthesised through condensation polymerization from the starting materials of 1,4-butanol and succinic acid, which was all derived from petrochemical process. Condensation polymerization of PBS requires succinic acid of higher purity (at least above 98%), which challenges the separation process of bio-succinic acid production. Effective recovery process of succinic acid need to be developed to fulfill the PBS synthesis using bio-succinic acid.
This study aims to provide an integrated process for biotechnological production of succinic acid which can be used for further PBS synthesis or chemical transformation. An effective separation process was developed and integrated to the fermentation by membrane unit to improve the succinic acid production. PBS was synthesized using succinic acid directly purified from its broth by the developed process in this study.
Section snippets
Bacterial strains, fermentation media and cultivation
E. coli MG-PYC (pTrchisA-pyc, △ldhA) was used for succinic acid fermentation. In this strain, the ldhA gene involved in the lactic acid synthesis pathway was deleted and heterologous pyruvate carboxylase (pTrchisA-pyc) was over-expressed for increased succinic acid production. Fermentation media contained per liter: 20 g glucose, 20 g tryptone, 10 g yeast extract, 0.15 g MgSO4, 0.2 g CaCl2, 0.02 g MnCl2, 0.45 g Na2HPO4·12H2O, 6 g NaH2PO4·2H2O and 3 g (NH4)2SO4·7H2O. Dual phase fed-batch fermentation
Fermentation
Fermentation experiment using glucose pulses was conducted in a bioreactor to examine the succinate production efficiency of this engineered E. coli. As shown in Fig. 2(A), succinate concentration reached 53 g/l, with considerable acetic acid and very little amount of formic acid and no lactic acid. Although the fermentation period is a little longer than 100 h with a 117 h anaerobic time, the yield is satisfactory with 1.07 mol succinate per mol glucose.
Further, succinic acid production in the
Conclusion
In this study, fermentation and separation was integrated by ultrafiltration membrane in this process. By this integration, the maximum succinic acid production was improved from 53 g/L (uncoupling at 129 h) to 73 g/L (coupling at 190 h). High purity succinic acid (99.4%) was obtained via this separation process with 90% recovery rate. Poly (butylene succinate) was first synthesized using succinic acid directly recovered from fermentation broth. This indicated that succinic acid recovered by this
Acknowledgements
We would like to thank Dr. Anders Thygesen from Technical University of Denmark for the language revision of this paper and the valuable suggestion.
This work was supported by the National Science Foundation of China (No. 21206175), the National High Technology Research and Development Program of China (863 Project, no. 2012AA101807, 2011AA02A203 and 2012AA022301).
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