Fermentative production of organic acids

The Industrial Biotechnology Group of Fraunhofer IGB develops processes for the production of platform chemicals using renewable raw materials. The focus is on the establishment, optimization and scaling of bioconversion processes in which biogenic raw materials are converted into basic chemicals either by microorganisms (bacteria, yeast or fungal cultures) or enzymes. Fungal systems have proven to be particularly advantageous for the microbial production of malic acid and itaconic acid.

Spectrum of services

• Selection and optimization of biocatalysts
• Development of suitable conversion processes on a laboratory scale
• Transfer of the conversion process to the pilot plant to m³ scale

Within process development, we look at all relevant parameters such as temperature, pO₂, OUR, CER and RQ or composition of the medium and establish perfectly adjusted mineral salt media and feeding strategies (repeated-batch, fed-batch or continuous culture management). On the basis of a statistical evaluation of all process parameters, we first transfer the optimal process from the shake flask to the fermenter (scale-over) and then into technical and pilot scale (scale-up). In addition, the downstream processing of products is enhanced.

To date, malic acid has been used primarily in the food and beverage industry. It improves the shelf-life of baked products and provides the sour taste of jams and juices. But it also boasts considerable potential as a building block in the chemical industry. Together with succinic and fumaric acid, it belongs to the group of C4 dicarboxylic acids. C4 acids can be converted into 1,4-butandiol (BDO) – an important precursor for further conversion into a wide variety of chemicals, including plastics, polymers and resins; the possible applications for these chemicals range from golf balls to printing inks and cleaning agents.

Fermentative production of malic acid was developed through the collaboration of the Industrial Biotechnology working group at Fraunhofer IGB and the Biotechnological Processes group at Fraunhofer CBP. Fermentation was carried out with the fungus Aspergillus oryzae, which is designated as a harmless food additive according to the GRAS (generally recognized as safe) status of the US Food and Drug Administration (FDA). In addition to glucose, the strain can also utilize the C5 sugar xylose, which is the main component of hemicellulose and thus can be sourced from wood residues.

Initially, the process was optimized at the laboratory scale; it was then established in stirred reactors and finally successfully scaled up to the 1 m³ scale using the substrate xylose for the first time. Downstream processing could be demonstrated using crystallization. In doing so, several kilograms of malic acid were produced that are now available as a sample for application tests.

Xylonic acid as a replacement for gluconic acid

Gluconic acid is an important constituent of foodstuffs, construction materials and dyes [1]. The acid is produced from glucose, which is obtained from plants rich in starch and thus competes with the production of foodstuffs. An alternative to gluconic acid is xylonic acid: on the one hand, this has similar properties and, on the other hand, it can be obtained from plant components containing lignocellulose or from agricultural waste material. The aim was therefore to develop an efficient process for obtaining xylonic acid from xylose.

250 g/L xylonic acid through optimization

The fermentation-based conversion of xylose is conducted using whole cell catalysis (Gluconobacter sp.), with addition of oxygen as a second reactant. In contrast to competing solutions, fermentation with Gluconobacter sp. has the advantage of being a specific, sustainable and efficient conversion. To date, the team of Industrial Biotechnology Group has achieved a xylonic acid concentration of over 250 g/L through optimization – with a yield of over 90 percent. In the subsequent rudimentary purification process, xylonic acid was obtained at a purity of over 80 percent, which is adequate for technical applications.

Scale-up and sample quantities for application-related investigations

The scalability of the process has already been demonstrated at the Fraunhofer Center for Chemical-Biotechnological Processes CBP by the team in the Biotechnological Processes Group with the 100-liter fermentation, a scale-up to 300 liters is planned. We are already making smaller quantities available for investigations for specific applications. For example, xylonic acid can be tested as a substitute for gluconic acid as a curing retardant for concrete or chelating agent.

Literature

[1] Toivari, M.H., Y. Nygard, M. Penttila, L. Ruohonen, and M.G. Wiebe, Microbial D-xylonate production. Applied Microbiology and Biotechnology, 2012. 96(1): p. 1-8.

Important synthesis building block 2,5-furandicarboxylic acid (FDCA)

2,5-furandicarboxylic acid (FDCA) and other dicarboxylic acids are important synthesis building blocks in the chemical industry due to their bifunctionality. 2,5-furandicarboxylic acid is also considered as a promising platform chemical with large market potential: the U.S. Department of Energy, for example, lists FDCA as one of the top 12 chemicals that can be produced from biomass. In a follow-up study, FDCA was also included in the top 10 due to its potential applications.

FDCA is considered so promising because of its high structural homology to terephthalic acid. Which is used to synthesize the widely used plastic polyethylene terephthalate (PET), whose annual market volume is approximately 40 million tons (2004 status).

Bio-based polyester PEF

Analogous to the polymerization of terephthalic acid to PET, FDCA can be polymerized to polyester polyethylene furanoate (PEF). FDCA, which can be produced from renewable resources, thus offers a sustainable substitute for petroleum-derived terephthalic acid in the production of bio-based polymers.

PEF is not only a bio-based plastic, but - unlike PET - it is also biodegradable. Haptically and visually, the polymer certainly resembles its petrochemical equivalent PET. PEF is not only characterized by its sustainability, but also exhibits significantly improved gas and water retention properties than PET: its water retention capacity is twice as high and it has six times and ten times better barrier properties to carbon dioxide and oxygen, respectively.

Biotechnological production of FDCA

FDCA can be produced from a variety of feedstocks, but hydroxymethylfurfural (HMF) is the most promising because it allows for different synthetic routes such as a chemical or biological synthesis (microbial or enzymatic). Biocatalysis represents a promising approach. Advantages include mild reaction conditions, lower cost, higher selectivity, and environmental friendliness. However, biological production of FDCA is not yet established.

Therefore, the research activities of Fraunhofer IGB focus on the establishment of whole cell catalysis of HMF to FDCA. In the BioConSept research project, we were able to successfully establish whole-cell microbial catalysis using targeted feeding strategies of hydroxymethylfurfural obtained from lignocellulosic biomass, achieving a yield of more than 80 percent at a concentration of up to 20 g/L FDCA in the laboratory. The subsequent scale-up was carried out using model experiments with scalable reactors or fermenters in the laboratory. Using dimensionless metrics selected in this process, we were able to design and successfully run the processes in the larger scale pilot plant.

The biotransformation of HMF to FDCA is also the subject of the current KEFIP research project. The aim of the project is to develop a sustainable multi-stage process for the conversion of inulin-containing chicory root beets, an agricultural waste product. HMF is extracted from inulin of the root beet by hydrothermal dehydration and subsequently oxidized to FDCA. The IGB is working in the project on the extraction of inulin from the root beet and also on the microbial oxidation of HMF to FDCA. In the field of FDCA production, the Fraunhofer IGB was able to show that a biotransformation of HMF to FDCA is possible when using an HMF solution from chicory which still contains various impurities. By optimizing the fermentation protocol it was possible that the yield of FDCA was exactly the same and there was no difference compared to the use of a pure HMF solution, which is free of impurities.

Furthermore, in the KEFIP project, the IGB developed a novel feeding strategy for the HMF solution based on online data from the fermentation.

The fermentative production process of itaconic acid dates back to 1932, at that time using Aspergillus itaconicus. Now, the Aspergillus terreus strain is primarily used. We were able to yield 137 g/L itaconic acid using A. terreus for biotechnological applications.

During the development of a furan dicarboxylic acid (FDCA) production process, we were able to successfully establish whole cell catalysis with Pseudomonas putida by adding hydroxymethylfurfural from biomass containing lignocellulose. In the laboratory, we were able to achieve a yield of more than 80 percent at a concentration up to 20 g/L FDCA through precise reaction control. The subsequent scale-up was carried out in model experiments with scalable reactors or fermenters in the laboratory. The dimensionless key figures selected in these experiments allowed us to adapt the processes to the larger scale of a pilot plant.