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

Accelerated Adaptive Laboratory Evolution by Automated Repeated Batch Processes in Parallelized Bioreactors

Adaptive laboratory evolution (ALE) is a valuable complementary tool for modern strain development. Insights from ALE experiments enable the improvement of microbial cell factories regarding the growth rate and substrate utilization, among others. Most ALE experiments are conducted by serial passaging, a method that involves large amounts of repetitive manual labor and comes with inherent experimental design flaws. The acquisition of meaningful and reliable process data is a burdensome task and is often undervalued and neglected, but also unfeasible in shake flask experiments due to technical limitations. Some of these limitations are alleviated by emerging automated ALE methods on the μL and mL scale. A novel approach to conducting ALE experiments is described that is faster and more efficient than previously used methods. The conventional shake flask approach was translated to a parallelized, L scale stirred-tank bioreactor system that runs controlled, automated, repeated batch processes. The method was validated with a growth optimization experiment of E. coli K-12 MG1655 grown with glycerol minimal media as a benchmark. Off-gas analysis enables the continuous estimation of the biomass concentration and growth rate using a black-box model based on first principles (soft sensor). The proposed method led to the same stable growth rates of E. coli with the non-native carbon source glycerol 9.4 times faster than the traditional manual approach with serial passaging in uncontrolled shake flasks and 3.6 times faster than an automated approach on the mL scale. Furthermore, it is shown that the cumulative number of cell divisions (CCD) alone is not a suitable timescale for measuring and comparing evolutionary progress

New miniature stirred-tank bioreactors for parallel study of enzymatic biomass hydrolysis

Many factors strongly influence the enzymatic hydrolysis of biomass to fermentable sugars (feedstock composition, pretreatment, enzymes and enzyme loading). In order to optimize the reaction conditions for the hydrolysis of biomass, an accurate high-throughput bioprocess development tool is mandatory, which enables a parallelization and an easy scale-up. New S-shaped impellers were developed for magnetically inductive driven stirred-tank bioreactors at a 10 mL-scale. An efficient and reproducible homogenization was shown at 20% w/w solids loading of microcrystalline cellulose and at, 4-10% with wheat straw in 48 parallel operated stirred-tank bioreactors. The scale-up was successfully validated for the enzymatic hydrolysis of wheat straw suspensions and microcrystalline cellulose mixtures by application of a cellulase complex at a milliliter- and liter-scale. As an example, the parallel stirred-tank bioreactor system was applied for the evaluation of enzymatic batch hydrolyses of plant materials with varying pretreatments