Clostridium Ljungdahlii Research Articles

Microbial electrosynthesis from carbon dioxide feedstock linked to yeast growth for the production of high-value isoprenoids

The difficulty in producing multi-carbon and thus high-value chemicals from CO2 is one of the key challenges of microbial electrosynthesis (MES) and other CO2 utilization technologies. Here, we demonstrate a two-stage bioproduction approach to produce terpenoids (>C20) and yeast biomass from CO2 by linking MES and yeast cultivation approaches. In the first stage, CO2 (C1) is converted to acetate (C2) using Clostridium ljungdahlii via MES. The acetate is then directly used as the feedstock to produce sclareol (C20), β-carotene (C40), and yeast biomass using Saccharomyces cerevisiae in the second stage. With the unpurified acetate-containing (1.5 g/L) spent medium from MES reactors, S. cerevisiae produced 0.32 ± 0.04 mg/L β-carotene, 2.54 ± 0.91 mg/L sclareol, and 369.66 ± 41.67 mg/L biomass. The primary economic analysis suggests that sclareol and biomass production is feasible using recombinant S. cerevisiae and non-recombinant S. cerevisiae, respectively, directly from unpurified acetate-containing spent medium of MES.

Methods of reaction and reactor engineering to adjust product ratios and increase efficiency of syngas fermentation with Clostridium ljungdahlii

Methods of reaction and reactor engineering to adjust product ratios and increase efficiency of syngas fermentation with <i>Clostridium ljungdahlii</i>

Clostridium ljungdahlii as a biocatalyst in microbial electrosynthesis – Effect of culture conditions on product formation

Microbial electrosynthesis enables the production of value-added chemicals from CO2 and electrons provided by an electrode. Clostridium ljungdahlii is an electroactive acetogen that potentially could be used in microbial electrosynthesis systems. However, the optimal operational parameters for microbial electrosynthesis using C. ljungdahlii are not known. Here, we explored the effects of yeast extract, pH, and cathode potential. A low initial pH increased the rate of acetate production from CO2 and H2 in serum bottle cultures. When cultivated in bioelectrochemical systems, the optimal coulombic efficiency (i.e. close to 100 %) was observed at a cathode potential between −0.8 V and −1.0 V, while the highest productivity was reached at −1.0 V. Addition of yeast extract to the medium was needed to ensure reproducible results. Using cyclic voltammetry, we detected hydrogen-mediated extracellular electron transfer of C. ljungdahlii during growth on CO2 in a bioelectrochemical system. These results show that operational parameters should be chosen carefully to maximise the efficiency of microbial electrosynthesis.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

BackgroundThe replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging.ResultsUsing a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol.ConclusionsWe thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Genetic Evidence Reveals the Indispensable Role of the rseC Gene for Autotrophy and the Importance of a Functional Electron Balance for Nitrate Reduction in Clostridium ljungdahlii.

For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: (1) the RNF complex-encoding gene cluster rnfCDGEAB; (2) the putative RNF regulator gene rseC; and (3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy, and in addition, contribute to understand the connection of nitrate reduction to energy metabolism.

Impact of different trace elements on metabolic routes during heterotrophic growth of C. ljungdahlii investigated through online measurement of the carbon dioxide transfer rate.

Synthesis 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.

13C-metabolic flux analysis of Clostridium ljungdahlii illuminates its core metabolism under mixotrophic culture conditions

Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO2 fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using 13C-metabolic flux analysis (13C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our 13C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. 13C-MFA during mixotrophic growth using the parallel tracers [1–13C]fructose, [1,2–13C]fructose, [1,2,3–13C]fructose, and [U–13C]asparagine found that externally supplied CO2 contributed the majority of carbon consumed. All internally-produced CO2 from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.

Newly explored formate dehydrogenases from Clostridium species catalyze carbon dioxide to formate

With concerns over global warming and climate change, many efforts have been devoted to mitigate atmospheric CO2 level. As a CO2 utilization strategy, formate dehydrogenase (FDH) from Clostridium species were explored to discover O2-tolerant and efficient FDHs that can catalyze CO2 to formate (i.e. CO2 reductase). With FDH from Clostridium ljungdahlii (ClFDH) that plays as a CO2 reductase previously reported as the reference, FDH from C.autoethanogenum (CaFDH), C. coskatii (CcFDH), and C. ragsdalei (CrFDH) were newly discovered via genome-mining. The FDHs were expressed in Escherichia coli and the recombinant FDHs successfully catalyzed CO2 reduction with a specific activity of 15 U g−1-CaFDH, 17 U g−1-CcFDH, and 8.7 U g−1-CrFDH. Interestingly, all FDHs newly discovered retain their catalytic activity under aerobic condition, although Clostridium species are strict anaerobe. The results discussed herein can contribute to biocatalytic CO2 utilization.

Effectively Converting Cane Molasses into 2,3-Butanediol Using Clostridiumljungdahlii by an Integrated Fermentation and Membrane Separation Process.

Firstly, 2,3-butanediol (2,3-BDO) is a chemical platform used in several applications. However, the pathogenic nature of its producers and the expensive feedstocks used limit its scale production. In this study, cane molasses was used for 2,3-BDO production by a nonpathogenic Clostridium ljungdahlii. It was found that cane molasses alone, without the addition of other ingredients, was favorable for use as the culture medium for 2,3-BDO production. Compared with the control (i.e., the modified DSMZ 879 medium), the differential genes are mainly involved in the pathways of carbohydrate metabolism, membrane transport, and amino acid metabolism in the case of the cane molasses alone. However, when cane molasses alone was used, cell growth was significantly inhibited by KCl in cane molasses. Similarly, a high concentration of sugars (i.e., above 35 g/L) can inhibit cell growth and 2,3-BDO production. More seriously, 2,3-BDO production was inhibited by itself. As a result, cane molasses alone with an initial 35 g/L total sugars was suitable for 2,3-BDO production in batch culture. Finally, an integrated fermentation and membrane separation process was developed to maintain high 2,3-BDO productivity of 0.46 g·L−1·h−1. Meanwhile, the varied fouling mechanism indicated that the fermentation properties changed significantly, especially for the cell properties. Therefore, the integrated fermentation and membrane separation process was favorable for 2,3-BDO production by C. ljungdahlii using cane molasses.

Recombinant expression and characterization of formate dehydrogenase from Clostridium ljungdahlii (ClFDH) as CO2 reductase for converting CO2 to formate

Recent concerns over climate change have triggered many efforts to mitigate atmospheric CO2 levels. Among the CO2 utilization strategies, we, herein, aimed to biochemically characterize formate dehydrogenase from Clostridium ljungdahlii (ClFDH) for bioconversion of CO2 to versatile formate. ClFDH was functionally expressed in Escherichia coli without any accessory proteins and recombinant ClFDH were successfully characterized as CO2 reductase with a specific activity of 9.1 mU mg−1 for CO2 reduction. Additionally, the optimum pH was determined at pH 7.0 and 9.0 for CO2 reduction and formate oxidation, respectively. In particular, ClFDH retain its catalytic activity under aerobic conditions, even if C. ljungdahlii is a representative strict anaerobic microorganism. The results discussed herein would not only contribute to broaden biological routes for CO2 utilization but also provide significant insight into the valorization of CO2-containing industrial waste gases to value-added chemicals.

Clostridium Ljungdahlii as a Biocatalyst in Microbial Electrosynthesis – Effect of Culture Conditions on Product Formation

Clostridium Ljungdahlii as a Biocatalyst in Microbial Electrosynthesis – Effect of Culture Conditions on Product Formation

Protein acetylation-mediated cross regulation of acetic acid and ethanol synthesis in the gas-fermenting Clostridium ljungdahlii

The autotrophic acetogen Clostridium ljungdahlii has emerged as a major candidate in the biological conversion of one-carbon gases (CO2/CO) to bulk chemicals and fuels. Nevertheless, the regulatory pathways and downstream metabolic changes responsible for product formation and distribution in this bacterium remain minimally explored. Protein lysine acetylation (PLA), a prevalent posttranslational modification, controls numerous crucial cellular functions. Herein, we revealed a novel cross-regulatory mechanism that uses both the PLA system and transcription factors to regulate the carbon flow distribution for product formation in C. ljungdahlii. The dominant acetylation/deacetylation system (At2/Dat1) in C. ljungdahlii was found to regulate the ratio of two major products, acetic acid and ethanol. Subsequent genetic and biochemical analyses revealed that the activities of Pta and AdhE1, two crucial enzymes responsible for acetic acid and ethanol synthesis, respectively, were greatly affected by their levels of PLA. We found that the acetylation statuses of Pta and AdhE1 underwent significant dynamic changes during the fermentation process, leading to differential synthesis of acetic acid and ethanol. Furthermore, the crucial redox-sensing protein Rex was shown to be regulated by PLA, which subsequently altered its transcriptional regulation on genes responsible for acetic acid and ethanol formation and distribution. Based on our understanding of this cross-regulatory module, we optimized the ethanol synthetic pathway by modifying the acetylation status (deacetylation-mimicked mutations of crucial lysine residues) of the related key enzyme, achieving significantly increased titer and yield of ethanol, an important chemical and fuel, by C. ljungdahlii in gas fermentation.

Design of Low-Cost Ethanol Production Medium from Syngas: An Optimization of Trace Metals for Clostridium ljungdahlii

Syngas fermentation via the Wood-Ljungdahl (WL) pathway is a promising approach for converting gaseous pollutants (CO and CO2) into high-value commodities. Because the WL involves several enzymes with trace metal components, it requires an adequate supply of micronutrients in the fermentation medium for targeted bioprocessing such as bioethanol production. Plackett-Burman statistical analysis was performed to examine the most efficient trace elements (Ni, Mg, Ca, Mn, Co, Cu, B, W, Zn, Fe, and Mo) and their concentrations for Clostridium ljungdahlii on ethanol production. Overall, 1.5 to 2.5 fold improvement in ethanol production could be achieved with designed trace element concentrations. The effects of tungsten and copper on ethanol and biomass production were determined to be the most significant, respectively. The model developed was statistically significant and has the potential to significantly decrease the cost of trace element solutions by 18–22%. This research demonstrates the critical importance of optimizing the medium for syngas fermentation in terms of product distribution and economic feasibility.

A Heterodimeric Reduced-Ferredoxin-Dependent Methylenetetrahydrofolate Reductase from Syngas-Fermenting Clostridium ljungdahlii.

ABSTRACTThe strict anaerobe Clostridium ljungdahlii can ferment CO or H2/CO2 via the Wood-Ljungdahl pathway to acetate, ethanol, and 2,3‐butanediol. This ability has attracted considerable interest, since it can be used for syngas fermentation to produce biofuels and biochemicals. However, the key enzyme methylenetetrahydrofolate reductase (MTHFR) in the Wood-Ljungdahl pathway of the strain has not been characterized, and its physiological electron donor is unclear. In this study, we purified the enzyme 46-fold with a benzyl viologen reduction activity of 41.2 U/mg from C. ljungdahlii cells grown on CO. It is composed of two subunits, MetF (31.5 kDa) and MetV (23.5 kDa), and has an apparent molecular mass of 62.2 kDa. The brownish yellow protein contains 0.73 flavin mononucleotide (FMN) and 7.4 Fe, in agreement with the prediction that MetF binds one flavin and MetV binds two [4Fe4S] clusters. It cannot use NAD(P)H as its electron donor or catalyze an electron-bifurcating reaction in combination with ferredoxin as an electron acceptor. The reduced recombinant ferredoxin, flavodoxin, and thioredoxin of C. ljungdahlii can serve as electron donors with specific activities of 91.2, 22.1, and 7.4 U/mg, respectively. The apparent Km values for reduced ferredoxin and flavodoxin were around 1.46 μM and 0.73 μM, respectively. Subunit composition and phylogenetic analysis showed that the enzyme from C. ljungdahlii belongs to MetFV-type MTHFR, which is a heterodimer, and uses reduced ferredoxin as its electron donor. Based on these results, we discuss the energy metabolism of C. ljungdahlii when it grows on CO or H2 plus CO2.IMPORTANCE Syngas, a mixture of CO, CO2, and H2, is the main component of steel mill waste gas and also can be generated by the gasification of biomass and urban domestic waste. Its fermentation to biofuels and biocommodities has attracted attention due to the economic and environmental benefits of this process. Clostridium ljungdahlii is one of the superior acetogens used in the technology. However, the biochemical mechanism of its gas fermentation via the Wood-Ljungdahl pathway is not completely clear. In this study, the key enzyme, methylenetetrahydrofolate reductase (MTHFR), was characterized and found to be a non-electron-bifurcating heterodimer with reduced ferredoxin as its electron donor, representing another example of MetFV-type MTHFR. The findings will form the basis for a deeper understanding of the energy metabolism of syngas fermentation by C. ljungdahlii, which is valuable for developing metabolic engineering strains and efficient syngas fermentation technologies.

Metabolic Engineering of Gas-Fermenting Clostridium ljungdahlii for Efficient Co-production of Isopropanol, 3-Hydroxybutyrate, and Ethanol.

Rational design and modification of autotrophic bacteria to efficiently produce high-value chemicals and biofuels are crucial for establishing a sustainable and economically viable process for one-carbon (C1) source utilization, which, however, remains a challenge in metabolic engineering. In this study, autotrophic Clostridium ljungdahlii was metabolically engineered to efficiently co-produce three important bulk chemicals, isopropanol, 3-hydroxybutyrate (3-HB), and ethanol (together, IHE), using syngas (CO2/CO). An artificial isopropanol-producing pathway was first constructed and optimized in C. ljungdahlii to achieve an efficient production of isopropanol and an unexpected product, 3-HB. Based on this finding, an endogenous active dehydrogenase capable of converting acetoacetate to 3-HB was identified in C. ljungdahlii, thereby revealing an efficient 3-HB-producing pathway. The engineered strain was further optimized to reassimilate acetic acid and synthesize 3-HB by introducing heterologous functional genes. Finally, the best-performing strain was able to produce 13.4, 3.0, and 28.4 g/L of isopropanol, 3-HB, and ethanol, respectively, in continuous gas fermentation. Therefore, this work represents remarkable progress in microbial production of bulk chemicals using C1 gases.

Functional dissection and modulation of the BirA protein for improved autotrophic growth of gas-fermenting Clostridiumljungdahlii.

SummaryGas‐fermenting Clostridium species can convert one‐carbon gases (CO2/CO) into a variety of chemicals and fuels, showing excellent application prospects in green biological manufacturing. The discovery of crucial genes and proteins with novel functions is important for understanding and further optimization of these autotrophic bacteria. Here, we report that the Clostridium ljungdahlii BirA protein (ClBirA) plays a pleiotropic regulator role, which, together with its biotin protein ligase (BPL) activity, enables an effective control of autotrophic growth of C. ljungdahlii. The structural modulation of ClBirA, combined with the in vivo and in vitro analyses, further reveals the action mechanism of ClBirA’s dual roles as well as their interaction in C. ljungdahlii. Importantly, an atypical, flexible architecture of the binding site was found to be employed by ClBirA in the regulation of a lot of essential pathway genes, thereby expanding BirA’s target genes to a broader range in clostridia. Based on these findings, molecular modification of ClBirA was performed, and an improved cellular performance of C. ljungdahlii was achieved in gas fermentation. This work reveals a previously unknown potent role of BirA in gas‐fermenting clostridia, providing new perspective for understanding and engineering these autotrophic bacteria.

Is reduced ferredoxin the physiological electron donor for MetVF-type methylenetetrahydrofolate reductases in acetogenesis? A hypothesis

The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.

Dynamic metabolic modelling predicts efficient acetogen-gut bacterium cocultures for CO-to-butyrate conversion.

While gas-fermenting acetogens have been engineered to secrete non-native metabolites such as butyrate, acetate remains the most thermodynamically favourable product. An alternative to metabolic engineering is to exploit native capabilities for CO-to-acetate conversion by coculturing an acetogen with a second bacterium that provides efficient acetate-butyrate conversion. We used dynamic metabolic modelling to computationally evaluate the CO-to-butyrate conversion capabilities of candidate coculture systems by exploiting the diversity of human gut bacteria for anaerobic synthesis of butyrate from acetate and ethanol. A preliminary screening procedure based on flux balance analysis was developed to identify 48 gut bacteria which satisfied minimal growth rate and acetate-to-butyrate conversion requirements when cultured on minimal medium containing acetate and a simple sugar not consumed by the paired acetogen. A total of 170 acetogen/gut bacterium/sugar combinations were dynamically simulated for continuous growth using a 70/30 CO/CO2 feed gas mixture and minimal medium computationally determined for each combination. While coculture systems involving the acetogens Eubacterium limosum or Blautia producta yielded low butyrate productivities and CO-to-ethanol conversion had minimal impact on system performance, dynamic simulations predicted a large number of promising coculture designs with Clostridium ljungdahlii or C. autoethanogenum as the CO-to-acetate converter. Pairings with the gut bacterium Clostridium hylemonae or Roseburia hominis were particularly promising due to their ability to generate high butyrate productivities over a range of dilution rates with a variety of sugars. The higher specific acetate secretion rate of C. ljungdahlii proved more beneficial than the elevated growth rate of C. autoethanogenum for coculture butyrate productivity. Our study demonstrated that metabolic modelling could provide useful insights into coculture design that can guide future experimental studies. More specifically, our predictions generated several favourable designs, which could serve as the first coculture systems realized experimentally.

Isobutanol Production by Autotrophic Acetogenic Bacteria.

Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.

Online monitoring of gas transfer rates during CO and CO/H2 gas fermentation in quasi-continuously ventilated shake flasks.

Syngas fermentation is a potential player for future emission reduction. The first demonstration and commercial plants have been successfully established. However, due to its novelty, development of syngas fermentation processes is still in its infancy, and the need to systematically unravel and understand further phenomena, such as substrate toxicity as well as gas transfer and uptake rates, still persists. This study describes a new online monitoring device based on the respiration activity monitoring system for cultivation of syngas fermenting microorganisms with gaseous substrates. The new device is designed to online monitor the carbon dioxide transfer rate (CO2 TR) and the gross gas transfer rate during cultivation. Online measured data are used for the calculation of the carbon monoxide transfer rate (COTR) and hydrogen transfer rate (H2 TR). In cultivation on pure CO and CO + H2 , CO was continuously limiting, whereas hydrogen, when present, was sufficiently available. The maximum COTR measured was approximately 5 mmol/L/h for pure CO cultivation, and approximately 6 mmol/L/h for cultivation with additional H2 in the gas supply. Additionally, calculation of the ratio of evolved carbon dioxide to consumed monoxide, similar to the respiratory quotient for aerobic fermentation, allows the prediction of whether acetate or ethanol is predominantly produced. Clostridium ljungdahlii, a model acetogen for syngas fermentation, was cultivated using only CO, and CO in combination with H2 . Online monitoring of the mentioned parameters revealed a metabolic shift in fermentation with sole CO, depending on COTR. The device presented herein allows fast process development, because crucial parameters for scale-up can be measured online in small-scale gas fermentation.