8 research outputs found
Characterization and optimization of 1-hexanol production in CO-utilizing clostridia
No full textThe goal of this thesis is the optimization of hexanol production from syngas. Hexanol can be used i.e.for the production of plastics, fuels and additives. Two potential biocatalysts, C. carboxidivorans and C. ljungdahlii, were characterized and evaluated for this application.These are the most important results obtained in this study: Bioenergetic calculations revealed that hexanol could be produced as the sole fermentation product from syngas. Hexanol toxicity was identified as a crucial limiting factor in hexanol production and could be circumvented by product extraction, increasing yields ~2.5 fold to 24 mM hexanol. The overall toxicity of hexanol towards both tested species was similar with acute inhibition at ≤ 20 mM. The hexanol tolerance of C. carboxidivorans was higher at 30°C (IC50 17.5 mM) than at 37°C (IC50 11.8 mM) and extraction led to detoxification only at the lower temperature. During scale-up from bottles to CSTR, gas delivery, measured as dissolved CO tension, and different nutrient requirements were identified as important bottlenecks. The highest hexanol titer produced in the CSTR was 37 mM. This is ~3 times the currently highest titer reported in other studies. To improve heterologous hexanol production with C.ljungdahlii, different enzymes were analyzed in transgenic C. ljungdahlii strains but the produced hexanol titers still remained lower than the titers obtained with C. carboxidivorans. In conjugation experiments with C. carboxidivorans, colonies could only be obtained either after heat shock of the recipient or after use of a donor strain expressing the C. carboxidivorans methyl transferases. These colonies displayed resistance towards the selection markers, but no verifiable liquid cultures could beobtained. The methylation pattern of C. carboxidivorans was investigated and the discovered motifs were attributed to respective methyl transferases. Converter gas (CG) was compared to synthetic CG with the same composition to investigate toxic effects of minor gas components. Without dilution of the gas and low cell densities, no growth was observed. High cell densities could partially circumvent inhibition and final product- and growth yields were similar with real and synthetic CG. If the gas phase was renewed, growth ceased even at high cell densities. No adaptation towards CG was observed. Compared to synthetic CG, hexanol production and growth yields were reduced with only H2+CO2 as substrate. Screening of different media compositions revealed improvement through addition of 2 g L-1 carbonate and a lower pH. In conclusion, C. carboxidivorans is a promising candidate for hexanol production from syngas. The hexanol titers obtained in this study of 37 mM (3.8 g L-1) is higher than titers reported for transgenic biocatalysts as well as co-cultivations. The major drawback is the lack of reliable methods for genetic manipulation. By addressing the crucial points identified in this work, hexanol production from syngas could be developed into a commercial process that can decrease net carbon emissions while simultaneously reducing dependence on fossil carbon sources. This is a step towards a greener andmore sustainable economy
Efficient whole cell biocatalyst for formate-based hydrogen production
No full textBackground: Molecular hydrogen (H2) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H2 could contribute significantly to the future energy mix. However, biological H2 production methods are faced with multiple barriers including substrate cost, low production rates, and low yields. The C1 compound formate is a promising substrate for biological H2 production, as it can be produced itself from various sources including electrochemical reduction of CO2 or from synthesis gas. Many microbes that can produce H2 from formate have been isolated; however, in most cases H2 production rates cannot compete with other H2 production methods.
Results: We established a formate-based H2 production method utilizing the acetogenic bacterium Acetobacterium woodii. This organism can use formate as sole energy and carbon source and possesses a novel enzyme complex, the hydrogen-dependent CO2 reductase that catalyzes oxidation of formate to H2 and CO2. Cell suspensions reached specific formate-dependent H2 production rates of 71 mmol g protein −1 h−1 (30.5 mmol g CDW −1 h−1) and maximum volumetric H2 evolution rates of 79 mmol L−1 h−1. Using growing cells in a two-step closed batch fermentation, specific H2 production rates reached 66 mmol g CDW −1 h−1 with a volumetric H2 evolution rate of 7.9 mmol L−1 h−1. Acetate was the major side product that decreased the H2 yield. We demonstrate that inhibition of the energy metabolism by addition of a sodium ionophore is suitable to completely abolish acetate formation. Under these conditions, yields up to 1 mol H2 per mol formate were achieved. The same ionophore can be used in cultures utilizing formate as specific switch from a growing phase to a H2 production phase.
Conclusions: Acetobacterium woodii reached one of the highest formate-dependent specific H2 productivity rates at ambient temperatures reported so far for an organism without genetic modification and converted the substrate exclusively to H2. This makes this organism a very promising candidate for sustainable H2 production and, because of the reversibility of the A. woodii enzyme, also a candidate for reversible H2 storage
Hexanol biosynthesis from syngas by Clostridium carboxidivorans P7 – product toxicity, temperature dependence and in situ extraction
No full textClostridium carboxidivorans converts syngas into industrial alcohols like hexanol, but titers may be limited by product toxicity. Investigation of IC50 at 30 °C (17.5 mM) and 37 °C (11.8 mM) revealed increased hexanol tolerance at lower temperatures. To avoid product toxicity, oleyl alcohol was added as an extraction solvent, increasing hexanol production nearly 2.5-fold to 23.9 mM (2.4 g/L) at 30 °C. This titer exceeds the concentration that is acutely toxic in the absence of a solvent, confirming the hypothesis that current hexanol production is limited by product toxicity. The solvent however had no positive effect at 37 °C. Furthermore, C. carboxidivorans cell membranes adapted to the higher temperature by incorporating more saturated fatty acids, but surprisingly not to hexanol. Corn oil and sunflower seed oil were tested as alternative, inexpensive extraction solvents. Hexanol titers were similar with all solvents, but oleyl alcohol achieved the highest extraction efficiency
Impact of different trace elements on metabolic routes during heterotrophic growth of C. ljungdahlii investigated through online measurement of the carbon dioxide transfer rate
No full textSynthesis gas fermentation using acetogenic clostridia is a rapidly increasing research area. It offers the possibility to produce platform chemicals from sustainable C1 carbon sources. The Wood-Ljungdahl pathway (WLP), which allows acetogens to grow autotrophically, is also active during heterotrophic growth. It acts as an electron sink and allows for the utilization of a wide variety of soluble substrates and increases ATP yields during heterotrophic growth. While glycolysis leads to CO2 evolution, WLP activity results in CO2 fixation. Thus, a reduction of net CO2 emissions during growth with sugars is an indicator of WLP activity. To study the effect of trace elements and ventilation rates on the interaction between glycolysis and the WLP, the model acetogen Clostridium ljungdahlii was cultivated in YTF medium, a complex medium generally employed for heterotrophic growth, with fructose as growth substrate. The recently reported anaRAMOS device was used for online measurement of metabolic activity, in form of CO2 evolution. The addition of multiple trace elements (iron, cobalt, manganese, zinc, nickel, copper, selenium, and tungsten) was tested, to study the interaction between glycolysis and the Wood ljungdahl pathway. While the addition of iron(II) increased growth rates and ethanol production, added nickel(II) increased WLP activity and acetate formation, reducing net CO2 production by 28%. Also, higher CO2 availability through reduced volumetric gas flow resulted in 25% reduction of CO2 evolution. These online metabolic data demonstrate that the anaRAMOS is a valuable tool in the investigation of metabolic responses i.e. to determine nutrient requirements that results in reduced CO2 production. Thereby the media composition can be optimized depending on the specific goal.All data is available upon request from the corresponding author
The Restriction–Modification Systems of Clostridium carboxidivorans P7
No full textClostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction–modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain
