CO2 as carbon source for microbial production of bio-based

Reducing waste and emissions of greenhouse gases like CO2 is a major demand for industry. In this context great interest has emerged in biological CO2-fixing processes which are supposed to be very effective in reducing CO2 emissions. Acetogenic bacteria are able to use hydrogen gas for the reduction CO2. The reductive acetyl-CoA pathway enables the autotrophic production of biobased chemicals like acetate, ethanol, butyrate, butanol, 2,3- butanediol, hexanoate and hexanol. Metabolic engineering of acetogens is a promising approach to enlarge the natural product portfolio. Low product yields and selectivities as well as low biomass densities and inefficient utilization of gaseous substrates are some of the challenges that slow down commercialization so far. The reaction engineering analyses of acetogens and the application of bioreactor designs providing high gas-liquid mass transfer efficiencies will enable new gas fermentation processes overcoming the challenges for further commercialization in the near future. Microalgae consuming the greenhouse gas CO2 and using sunlight as energy source could become an important renewable source for biobased chemicals. Since the production cost of most microalgae products from current mass cultivation systems is still prohibitively high, further development is required. To advance economic microalgae mass production new open thin-layer cascade photobioreactors were designed recently up to a pilot scale (50 m2) for high-cell density cultivation of saline microalgae achieving up to 50 g L−1 dry cell mass, which was shown applying physically simulated climate conditions of a Mediterranean summer in Spain in the TUM-AlgaeTec-Center near by Munich, Germany. Finally, state-of-the-art of phototrophic CO2-fixation by microalgae and of autotrophic CO2-fixation by acetogens will be compared with respect to kinetics, process engineering aspects and productivities. References Riegler P, Chrusciel T, Mayer A, Doll K, Weuster Botz D (2019): Reversible retrofitting of a stirred-tank bioreactor for gas-lift operation to perform syngas fermentation studies. Biochem Eng J 141: 89-101. Doll K, Rückel A, Kaempf P, Weuster-Botz D (2018): Two stirred-tank bioreactors in series enable continuous production of alcohols from carbon monoxide with Clostridium carboxidivorans. Bioproc Biosys Eng 41:1403- 1416. Severin TS, Apel A, Brueck T, Weuster-Botz D (2018): Investigation of vertical mixing in open thin-layer cascade reactors using Computational Fluid Dynamics. Chem Eng Res Design 132: 436-444. Apel AC, Pfaffinger CE, Basedahl N, Mittwollen N, Goebel J, Sauter J, Brueck T, Weuster-Botz D (2017): Open thin-layer cascade reactors for saline microalgae production evaluated in a physically simulated Mediterranean summer climate. Algal Research 25 (2017): 381–390. Koller A, Loewe H, Schmid V, Mundt S, Weuster-Botz D (2017) Model-supported phototrophic growth studies with Scenedesmus obtusiusculus in a flat-plate photobioreactor. Biotechnol Bioeng 114: 308–320 Groher A, Weuster-Botz D (2016): Comparative reaction engineering analysis of different acetogenic bacteria for gas fermentation. J Biotechnol 228: 82-94. Kantzow C, Mayer A, Weuster-Botz D (2015): Continuous gas fermentation by Acetobacterium woodii in a submerged membrane reactor with full cell retention. J Biotechnol 212: 11-18

Anodic respiration of Pseudomonas putida KT2440 in a stirred-tank bioreactor

Anodic batch production of para-hydroxybenzoic acid (pHBA) from citric acid with a genetically modified Pseudomonas putida KT2440 strain was studied in a bio-electrochemical system (BES) based on a standard lab-scale stirred-tank bioreactor at fully controlled anaerobic reaction conditions. Electron transfer to the anode was mediated by addition of KFe(CN) to the medium. Effects of varying anode surface areas (graphite rod, felt and brush), power input (stirrer speed) and mediator concentrations were investigated. The obligate aerobic P. putida grew anaerobically with mediated anodic respiration and pHBA production was observed. Anodic respiration was best applying the graphite rod electrode which showed a maximal current density of 12.5 mA cm. This is the highest measured for non-porous electrodes in BES until now. Increasing the power input to 2.93 W L (700 rpm) and online control of the redox potential E at 225 mV (vs. Ag/AgCl) in the medium by controlled addition of mediator resulted in a maximal pHBA yield of 9.91 mmolC molC which exceeds pHBA yields in the aerobic batch process by 69 % (5.87 mmolC molC )

Asymmetric Whole-Cell Bio-Reductions of (R)-Carvone Using Optimized Ene Reductases

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E. coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts’ activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E. coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature

Miniaturization in Biocatalysis

The use of biocatalysts for the production of both consumer goods and building blocks for chemical synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technology and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chemicals, intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Background Conventional experiments in small scale are often performed in a Black Box fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. Results The same mastermix (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). Conclusions The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities