EVALUATION OF RENEWABLE RESOURCES AS CARBON SOURCES FOR ORGANIC ACID PRODUCTION WITH FILAMENTOUS FUNGI zur Erlangung des akademischen Grades eines DOKTORS DER INGENIEURWISSENSCHAFTEN (Dr.-Ing.) der Fakultät für Chemieingenieurwesen und Verfahrenstechnik des Karlsruher Instituts für Technologie (KIT) genehmigte DISSERTATION von M. Sc. Stefan Dörsam aus Weinheim, Baden-Württemberg Referent: Prof. Dr. Christoph Syldatk Korreferent: Prof. Dr. Nicolaus Dahmen Tag der mündlichen Prüfung: 16.03.2018 Preamble Parts of this thesis are based on peer reviewed research articles. All articles have been drafted during this work and describe the major results of the evaluation of renewable resources for malic and fumaric acid production with the filamentous fungi A. oryzae and R. delemar. Chap- ters based on previous published work are indicated as such at the beginning of the chapter. The text of these chapters is partly identical to the content of the publications. Layout, citation style, figures and formatting have been modified and adjusted to the style of this dissertation. i List of Publications Peer reviewed original Publications: Dörsam S., Fesseler J., Gorte O., Hahn T., Zibek S., Syldatk C. and Ochsenreither K. (2017). “Sustainable Carbon Sources for Microbial Organic Acid Production with Filamentous Fungi,” Biotechnology for Biofuels 10 (1). BioMed Central: 242. doi:10.1186/s13068-017- 0930-x. Dörsam S., Kirchoff J., Bigalke M., Dahmen N., Syldatk C. and Ochsenreither, K. (2016). “Evaluation of Pyrolysis Oil as Carbon Source for Fungal Fermentation,” Frontiers in Microbiology 7 (December): 1–11. doi:10.3389/fmicb.2016.02059 Oswald F.*, Dörsam S.*, Veith N., Zwick M., Neumann A., Ochsenreither K. and Syldatk, C. (2016). “Sequential Mixed Cultures: From Syngas to Malic Acid.” Frontiers in Microbiology 7 (June): 1–12. doi:10.3389/fmicb.2016.00891 *Co-first authorship Book Chapter Neumann A., Dörsam S., Oswald F., Ochsenreither K. (2016) Microbial Production of Value- Added Chemicals from Pyrolysis Oil and Syngas. In: Xian M. (eds) Sustainable Produc- tion of Bulk Chemicals. Springer, Dordrecht: 69–105. doi:10.1007/978-94-017-7475-8_4 Conference Poster Dörsam, S., Syldatk, C. and Ochsenreither, K. (2017). “Evaluation of renewable resources for fungal organic acid production,” 2. Bioökonomiekongress Baden-Württemberg Dörsam S., Syldatk C. and Ochsenreither, K. (2016). “Toxic effects of different substances from pyrolysis oil on growth and production of malic acid by Aspergillus oryzae,” VAAM Jahrestagung Dörsam S., Syldatk C. and Ochsenreither K. (2015). “Malate production from renewable re- sources by Aspergillus oryzae,” FEMS, 6th Congress of European Microbiologists Dörsam S., Syldatk C. and Ochsenreither, K. (2014). “Evaluation of pyrolysis oil as platform for fungal fermentation,” 1. Bioökonomiekongress Baden-Württemberg ii Abstract Abstract The transformation of the modern economy to a bio-economy, based on biological raw materi- als requires completely new approaches in research, development and production. Above all, the combination of biotechnological and chemical substance conversion plays an important role. While developing processes based on renewable resources, the “food or fuel” dilemma has to be strongly considered. Lignocellulose as feedstock for those kinds of processes could be a part of the solution for this problem. The lignocellulosic bio refinery supplies pretreated frac- tions of lignocellulose, accessible for chemistry and biotechnology that possibly can be metab- olized and converted by microorganisms to more valuable chemicals and platform chemicals. Promising candidates for these demands are dicarboxylic acids. Because of the high diversity of convertible carbon sources, as well as the known robustness fungi are the optimal organic acid producer used for the evaluation of renewable resources as carbon sources for organic acid production. In addition to L-malate, fumarate is one of the most important, high-quality basic chemical that can be produced from renewable resources through microbial fermentation. Two different pretreatment methods of lignocellulose for fermentation were evaluated in this work. The pyrolysis process leads to two fractions, an organic (pyrolysis oil) and an aqueous condensate. Further gasification of a mixture of both condensates leads to the formation of syngas. During the organosolv-process, lignocellulose is separated into its basic components, lignin, cellulose and hemicellulose. In secondary refining, saccharification of the cellulose into glucose and hemicellulose into xylose is carried out by enzymatic hydrolysis. Pyrolysis oil, a complex mixture of several organic compounds, produced during flash pyrolysis of organic lignocellulosic material was evaluated for its suitability as alternative carbon source for fungal growth and fermentation processes (chapter 2). Therefore several fungi from all phyla were screened for their tolerance towards pyrolysis oil. Additionally Aspergillus oryzae and Rhizopus delemar, both established organic acid producers, were chosen as model organisms to investigate the suitability of pyrolysis oil as carbon source in fungal production processes. It was observed that A. oryzae tolerates pyrolysis oil concentrations between 1 - 2 % depending on growth phase or stationary production phase, respectively. To investigate possible reasons for the low tolerance level, eleven compounds from pyrolysis oil representing aldehydes, or- ganic acids, small organic compounds and phenolic substances were selected to determine max- imum concentrations still allowing growth and organic acid production. Furthermore, the effect iii Abstract of various substances to malic acid production were analyzed and compounds were categorized regarding their properties. To validate the results, further tests were also performed with R. delemar. For the first time it could be shown that small amounts of phenolic substances are even beneficial for organic acid production and A. oryzae might be able to degrade isoeugenol. Regarding pyrolysis oil toxicity, 2-cyclopenten-1-on was identified as the most toxic compound for filamentous fungi. This compound has never been described for anti-fungal or any other toxic properties before and possibly is responsible for the low fungal tolerance levels towards pyrolysis oil. The aqueous condensate produced during flash pyrolysis of organic lignocellulosic material, was analyzed as substrate in the third chapter of this work. The main components are acetic acid with 4.5%, hydroxyacetone with 3.5%, methanol with 1.7%, ethylene glycol with 0.5% and propionic acid with 0.4%. As the effect of methanol in main culture medium in this con- centration range is minimal, hydroxyacetone is the only main component with an inhibitory concentration below the concentration in the aqueous condensate. The cultivation in aqueous condensate model mixtures showed the possible suitability of the aqueous condensate when diluted 1:1. For evaluating the aqueous condensate as carbon source, shake flask cultivations with diluted and undiluted fractions, as well as several detoxifying pretreatments done to reduce the amount of phenolic and solvent like compounds but did not result in product formation. The fourth chapter of this thesis describes the evaluation of synthesis gas (Syngas) as a possible carbon source for fermentation with the filamentous fungus A. orzae. Because this fungus is not able to metabolize sygase directly, the idea of a coupled process in which one organism pro- duces a product from syngas which can also be the carbon source for the fungus was imple- mented. Synthesis gas fermentation using acetogenic bacteria is an approach for production of bulk chemicals like acetate, ethanol, butanol or 2,3-butandiol avoiding the “food or fuel” di- lemma by using carbon monoxide, carbon dioxide and hydrogen from gasification of biomass or industrial waste gases. In this study, it was shown that Aspergillus oryzae is able to produce malic acid using acetate as sole carbon source which is a main product of acetogenic syngas fermentation. During the syngas fermentation as part of the sequential mixed culture, Clostrid- ium ljungdahlii was grown with artificial syngas modeling a composition of clean syngas from entrained bed gasification of straw (32.5 vol-% CO, 32.5 vol-% H2, 16 vol-% CO2 and 19 vol- % N2). Syngas consumption was monitored via automated gas chromatographic measurement of the off-gas. For the sequential fungal fermentation part gas sparging was switched from syn- gas to 0.6 L/min of air. Ammonia content of medium for syngas fermentation was reduced to iv Abstract 0.33 g/L NH4Cl to meet the requirements for fungal production of dicarboxylic acids. Malic acid production performance of A. oryzae in organic acid production medium and syngas me- dium with acetate as sole carbon source was verified and gave YP/S values of 0.28 g/g and 0.37 g/g respectively. Growth and acetate formation of C. ljungdahlii during syngas fermenta- tion were not affected by the reduced ammonia content and 66 % of the consumed syngas was converted to acetate. The overall conversion of CO and H2 into malic acid was calculated to be 3.5 g malic acid per mol of consumed syngas or 0.22 g malic acid per gram of syngas. The fifth chapter of this work describes the evaluation of the second pretreatment method for lignocellulose used in this study. The organic acid producer A. oryzae and R. delemar are able to convert several alternative carbon sources to malic and fumaric acid.