Clostridium Ljungdahlii Research Articles

Clostridia as Promising Biofactory for Heterotrophic (Fructose) and Autotrophic (C1-Gas) Fermentation

This study compared the performance of Clostridium ljungdahlii and Clostridium aceticum in the fermentation of fructose and C1-gasses (CO, CO2, N2) to produce valuable products such as ethanol and acetic acid. In heterotrophic fermentation (fructose), C. ljungdahlii yielded high ethanol concentrations (350 mg/L) and acetic acid (500 mg/L), with optimal production at pH 8 on the first day of fermentation. Although autotrophic fermentation (C1-gasses) resulted in lower ethanol levels (200 mg/L), it remained a viable option. Conversely, C. aceticum predominantly produced acetic acid in both fermentation modes, with higher concentrations in the heterotrophic fermentation (1600 mg/L) than the autotrophic fermentation (380 mg/L). These findings demonstrate the versatility of both microorganisms for producing valuable metabolites. C. ljungdahlii shows promise for bioethanol production, while C. aceticum excels at generating acetic acid, a crucial component in bioplastics and various industrial processes.

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures.

Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, "state-of-the-art" labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum, C. ljungdahlii, and Clostridium kluyveri. We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Power-to-Vitamins: producing folate (vitamin B9) from renewable electric power and CO2 with a microbial protein system

We recently proposed a two-stage Power-to-Protein technology to produce microbial protein from renewable electric power and CO2. Two stages were operated in series: Clostridium ljungdahlii in Stage A to reduce CO2 with H2 into acetate, and Saccharomyces cerevisiae in Stage B to utilize O2 and produce microbial protein from acetate. Renewable energy can be used to power water electrolysis to produce H2 and O2. A drawback of Stage A was the need for continuous vitamin supplementation. In this study, by using the more robust thermophilic acetogen Thermoanaerobacter kivui instead of C. ljungdahlii, vitamin supplementation was no longer needed. Additionally, S. cerevisiae produced folate when grown with acetate as a sole carbon source, achieving a total folate concentration of 6.7 mg per 100 g biomass with an average biomass concentration of 3 g l-1. The developed Power-to-Vitamin system enables folate production from renewable power and CO2 with zero or negative net-carbon emissions.

Pooled CRISPR Interference Screening Identifies Crucial Transcription Factors in Gas-Fermenting Clostridium ljungdahlii.

Gas-fermenting Clostridium species hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) in Clostridium ljungdahlii, a representative species of gas-fermenting Clostridium, with a special focus on TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth of C. ljungdahlii, respectively. We focused our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleoside triphosphates (dNTPs) supply, carbon fixation, and product synthesis in C. ljungdahlii, thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression of deoR in C. ljungdahlii by adjusting its promoter strength, leading to an improved growth rate and ethanol synthesis of C. ljungdahlii when utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermenting Clostridium species, expanding the horizons for functional genomic research in these industrially important bacteria.

ZnMo-MOF as anti-CO hydrogen electrocatalyst enhance microbial electrosynthesis for CO/CO2 conversion

Microbial electrosynthesis (MES) is an electrically driven technology that can be used for converting CO/CO2 into chemicals. The unique electronic and substrate properties of CO make it an important research target for MES. However, CO can poison the cathode and increase the overpotential of hydrogen evolution reaction (HER), thus reducing the electron transfer rate via H2. This work evaluated the effect of an anti-CO HER catalyst on the performance of MES for CO/CO2 conversion. ZnMo-metal–organic framework (MOF) materials with different calcination temperatures were synthesized. ZnMo-MOF-800 with Mo2C nanoparticles as active centers exhibited excellent resistance to CO toxicity. It also obtained the highest hydrogen evolution and enhanced electron transfer rate in CO atmosphere. MES with ZnMo-MOF-800 cathode and Clostridium ljungdahlii as biocatalyst obtained 0.31 g L−1 d−1 acetate yield, 0.1 g L−1 d−1 butyrate yield, and 0.09 g L−1 d−1 2,3-butanediol yield in CO/CO2, while Pt/C only get 0.076 g L−1 d−1 acetate yield, 0.05 g L−1 d−1 butyrate yield and 0.02 g L−1 d−1 2,3-butanediol yield. ZnMo-MOF-800 was conducive to biofilm formation, enabling it to better resist CO toxicity. This work provides new opportunities for constructing a highly efficient cathode with an anti-CO hydrogen evolution catalyst to enhance CO/CO2 conversion in MES.

Bioelectricity and CO2-to-butyrate production using photobioelectrochemical cells with bio-hydrogel

Bio-photoelectrochemical cell (BPEC) is an emerging technology that can convert the solar energy into electricity or chemicals. However, traditional BPEC depending on abiotic electrodes is challenging for microbial/enzymatic catalysis because of the inefficient electron exchange. Here, electroactive bacteria (Shewanella loihica PV-4) were used to reduce graphene oxide (rGO) nanosheets and produce co-assembled rGO/Shewanella biohydrogel as a basic electrode. By adsorbing chlorophyll contained thylakoid membrane, this biohydrogel was fabricated as a photoanode that delivered maximum photocurrent 126 μA/cm3 under visible light. Impressively, the biohydrogel could be served as a cathode in BPEC by forming coculture system with genetically edited Clostridium ljungdahlii. Under illumination, the BPEC with above photoanode and cathode yielded ∼ 5.4 mM butyrate from CO2 reduction, 169 % increase compared to dark process. This work provided a new strategy (nanotechnology combined with synthetic biology) to achieve efficient bioelectricity and valuable chemical production in PBEC.

Electrobiocorrosion by microbes without outer-surface cytochromes.

Anaerobic microbial corrosion of iron-containing metals causes extensive economic damage. Some microbes are capable of direct metal-to-microbe electron transfer (electrobiocorrosion), but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation. Previous studies have suggested that respiration with 316L stainless steel as the electron donor is indicative of electrobiocorrosion, because, unlike pure Fe0, 316L stainless steel does not abiotically generate H2 as an intermediary electron carrier. Here, we report that all of the methanogens (Methanosarcina vacuolata, Methanothrix soehngenii, and Methanobacterium strain IM1) and acetogens (Sporomusa ovata and Clostridium ljungdahlii) evaluated respired with pure Fe0 as the electron donor, but only M. vacuolata, Mx. soehngenii, and S. ovata were capable of stainless steel electrobiocorrosion. The electrobiocorrosive methanogens required acetate as an additional energy source in order to produce methane from stainless steel. Cocultures of S. ovata and Mx. soehngenii demonstrated how acetogens can provide acetate to methanogens during corrosion. Not only was Methanobacterium strain IM1 not capable of electrobiocorrosion, but it also did not accept electrons from Geobacter metallireducens, an effective electron-donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe0. The finding that M. vacuolata, Mx. soehngenii, and S. ovata are capable of electrobiocorrosion, despite a lack of the outer-surface c-type cytochromes previously found to be important in other electrobiocorrosive microbes, demonstrates that there are multiple microbial strategies for making electrical contact with Fe0.

Understanding microbiome dynamics and functional responses during acidogenic fermentation of sucrose and sugarcane vinasse through metatranscriptomic analysis

Improving anaerobic digestion of sugarcane vinasse – a high-strength wastewater from ethanol distillation – is a subject of great interest, in view of the reduction of the pollutants and recovery of methane and valuable metabolites as byproducts. Through metatranscriptomic analysis, this study evaluated the active microbiome and metabolic pathways in a continuous acidogenic reactor: Stage 1S (control): 100% sucrose-based substrate (SBS); Stage 2SV (acclimation): 50% SBS and 50% vinasse; Stage 3V: 100% vinasse. Metatranscriptome obtained from each Stage was subjected to taxonomic and functional annotations. Under SBS feeding, pH dropped to pH 2.7 and biohydrogen production was observed. As vinasse was added, pH increased to 4.1–4.5, resulting in community structure and metabolite changes. In Stage 3V, biohydrogen production ceased, and propionate and acetate prevailed among the volatile fatty acids. Release of homoacetogenesis enzymes by Clostridium ljungdahlii and of uptake hydrogenase (EC 1.12.99.6) by Pectinatus frisingensis were linked to hydrogen consumption in Stages 2SV and 3V. Metabolic pathways of vinasse compounds, such as carbohydrates, malate, oxalate, glycerol, sulfate and phenol, were investigated in detail. In pyruvate metabolism, gene transcripts of oadA (oxaloacetate decarboxylase) and mdh (malate dehydrogenase), were upregulated in Stage 3V, being mostly attributed to P. frisingensis. Acetate formation from vinasse degradation was mainly attributed to Megasphaera and Clostridium, and propionate formation to P. frisingensis. Glycerol removal from vinasse exceeded 99%, and gene transcripts encoding for glpF (glycerol uptake facilitator protein), glpK (glycerol kinase) and glpABC (glycerol-3-phosphate dehydrogenase) were expressed mostly by Pectinatus and Prevotella. mRNA profiling showed that active bacteria and gene expression greatly changed when vinasse replaced sucrose, and Pectinatus was the main active bacterium degrading the searched compounds from vinasse. The identification of the main metabolic routes and the associated microorganisms achieved in this work contributes with valuable information to support further optimization of fermentation towards the desired metabolites.

Impact of cathodic pH and bioaugmentation on acetate and CH4 production in a microbial electrosynthesis cell.

This study compares carbon dioxide conversion in carbonate-fed microbial electrosynthesis (MES) cells operated at low (5.3), neutral (7) and high (8) pH levels and inoculated either with wild-type or bioaugmented mixed microbial populations. Two 100 mL (cathode volume) MES cells inoculated with anaerobic digester sludge were operated with a continuous supply of carbonate solution (5 g L-1 as CO3 2-). Acetate production was highest at low pH, however CH4 production still persisted, possibly due to pH gradients within the cathodic biofilm, resulting in acetate and CH4 volumetric (per cathode compartment volume) production rates of 1.0 ± 0.1 g (Lc d)-1 and 0.84 ± 0.05 L (Lc d)-1, respectively. To enhance production of carboxylic acids, four strains of acetogenic bacteria (Clostridium carboxidivorans, Clostridium ljungdahlii, Clostridium autoethanogenum, and Eubacterium limosum) were added to both MES cells. In the bioaugmented MES cells, acetate production increased to 2.0 g (Lc d)-1. However, production of other carboxylic acids such as butyrate and caproate was insignificant. Furthermore, 16S rRNA gene sequencing of cathodic biofilm and suspended biomass suggested a low density of introduced acetogenic bacteria implying that selective pressure rather than bioaugmentation led to improved acetate production.

Novel experimental data-driven bubble breakage model for universal application in Euler-Lagrange multiphase frameworks

An experimental data driven model for the prediction of bubble breakage in gas–liquid multiphase reactors was developed which fulfills requirements of the Euler-Lagrangian (EL) framework for the analysis of individual bubbles. The critical Weber number (Wecrit) serves as the breakage criterion in combination with the minimum time between two consecutive breakage events. Wecrit indicates when the hydrodynamic forces acting on an individual bubble surmount its surface tension. Once the threshold is passed a binary bubble breakup event occurs, which results in the formation of daughter bubbles assigned to an M−shaped daughter size distribution (DSD). Optimum Wecrit values were obtained by minimizing the difference between simulated and measured Sauter mean diameters d32. The latter were derived from bubble size distributions (BSDs) measurements that cover about 11 – 89 W m−3 power inputs. Using deionized water and a minimum timespan of 30 ms between two consecutive breakages Wecrit = 6.1 was determined. To extend the scope of the model, characteristic media compositions for so-called syngas fermentations with Clostridium ljungdahlii were investigated. To match surface tension and viscosity with those of fresh medium (FM) and late phase medium (LPM), two aqueous solutions containing sodium dodecyl sulfate (SDS) and glycerol were prepared. Notably, the breakage model managed to well predict the impact of altered fluid properties of FM and LPM without requiring further calibration. The sum of Sauter diameter deviation between simulations and experiments was only 0.21 and 0.40 mm for FM and LPM, respectively.

Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting Clostridium ljungdahlii.

Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO2/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, we identified a RpiR family transcription factor (TF) that can regulate the autotrophic growth and carbon fixation of C. ljungdahlii. After deletion of the corresponding gene, the resulting mutant strain exhibited significantly impaired growth in gas fermentation, thus reducing the production of acetic acid and ethanol. In contrast, the overexpression of this TF gene could promote cell growth, indicating a positive regulatory effect of this TF in C. ljungdahlii. Thus, we named the TF as GssR (growth and solvent synthesis regulator). Through the following comparative transcriptomic analysis and biochemical verification, we discovered three important genes (encoding pyruvate carboxylase, carbon hunger protein CstA, and a BlaI family transcription factor) that were directly regulated by GssR. Furthermore, an upstream regulator, BirA, that could directly bind to gssR was found; thus, these two regulators may form a cascade regulation and jointly affect the physiology and metabolism of C. ljungdahlii. These findings substantively expand our understanding on the metabolic regulation of carbon fixation in gas-fermenting Clostridium species.

Self-assembly of graphene oxide and Shewanella oneidensis MR-1 formed a conductive bio-abiotic composite for enhancing microbial electrosynthesis performance

Microbial electrosynthesis (MES) is an attractive technology for converting CO2 into chemicals. In this technique, electrons are acquired either directly or indirectly through H2 in the cathode. Shewanella oneidensis MR-1 can perform hydrogen evolution reaction via reverse electron transfer at the cathode. In this study, the self-assembly of graphene oxide and S. oneidensis MR-1 formed a conductive bio-abiotic composite as a cathode for MES with Clostridium ljungdahlii, obtaining more biocatalysts for hydrogen evolution and enhancing electron transfer rate. The average H2 production rate with the conductive bio-abiotic composite was 41.51 ± 2.54 × 102 mol d−1, while the undecorated carbon felt was 12.96 ± 1.09 × 102 mol d−1. Consequently, acetate and butyrate yields were 0.18 g L−1 d−1 and 0.07 g L−1 d−1, which increased by 2.1 and 1.7 times those of the control, respectively. This work provides new opportunities for constructing a highly efficient cathode via biotic-abiotic hybrid composite for improving MES performance.

Thermodynamic and Kinetic Modeling Directs Pathway Optimization for Isopropanol Production in a Gas-Fermenting Bacterium.

Rational engineering of gas-fermenting bacteria for high yields of bioproducts is vital for a sustainable bioeconomy. It will allow the microbial chassis to renewably valorize natural resources from carbon oxides, hydrogen, and/or lignocellulosic feedstocks more efficiently. To date, rational design of gas-fermenting bacteria such as changing the expression levels of individual enzymes to obtain the desired pathway flux is challenging, because pathway design must follow a verifiable metabolic blueprint indicating where interventions should be executed. Based on recent advances in constraint-based thermodynamic and kinetic models, we identify key enzymes in the gas-fermenting acetogen Clostridium ljungdahlii that correlate with the production of isopropanol. To this extent, we integrated a metabolic model in comparison with proteomics measurements and quantified the uncertainty for a variety of pathway targets needed to improve the bioproduction of isopropanol. Based on in silico thermodynamic optimization, minimal protein requirement analysis, and ensemble modeling-based robustness analysis, we identified the top two significant flux control sites, i.e., acetoacetyl-coenzyme A (CoA) transferase (AACT) and acetoacetate decarboxylase (AADC), overexpression of which could lead to increased isopropanol production. Our predictions directed iterative pathway construction, which enabled a 2.8-fold increase in isopropanol production compared to the initial version. The engineered strain was further tested under gas-fermenting mixotrophic conditions, where more than 4 g/L isopropanol was produced when CO, CO2, and fructose were provided as the substrates. In a bioreactor environment sparging with CO, CO2, and H2 only, the strain produced 2.4 g/L isopropanol. Our work highlighted that the gas-fermenting chasses can be fine-tuned for high-yield bioproduction by directed and elaborative pathway engineering. IMPORTANCE Highly efficient bioproduction from gaseous substrates (e.g., hydrogen and carbon oxides) will require systematic optimization of the host microbes. To date, the rational redesign of gas-fermenting bacteria is still in its infancy, due in part to the lack of quantitative and precise metabolic knowledge that can direct strain engineering. Here, we provide a case study by engineering isopropanol production in gas-fermenting Clostridium ljungdahlii. We demonstrate that a modeling approach based on the thermodynamic and kinetic analysis at the pathway level can provide actionable insights into strain engineering for optimal bioproduction. This approach may pave the way for iterative microbe redesign for the conversion of renewable gaseous feedstocks.

Direct detection of a single [4Fe‐4S] cluster in a tungsten‐containing enzyme: Electrochemical conversion of CO2 into formate by formate dehydrogenase

AbstractThe conversion of CO2 into fuels and valuable chemicals is one of the central topics to combat climate change and meet the growing demand for renewable energy. Herein, we show that the formate dehydrogenase from Clostridium ljungdahlii (ClFDH) adsorbed on electrodes displays clear characteristic voltammetric signals that can be assigned to the reduction and oxidation potential of the [4Fe‐4S]2+/+ cluster under nonturnover conditions. Upon adding substrates, the signals transform into a specific redox center that engages in catalytic electron transport. ClFDH catalyzes rapid and efficient reversible interconversion between CO2 and formate in the presence of substrates. The turnover frequency of electrochemical CO2 reduction is determined as 1210 s−1 at 25 °C and pH 7.0, which can be further enhanced up to 1786 s−1 at 50°C. The Faradaic efficiency at −0.6 V (vs. standard hydrogen electrode) is recorded as 99.3% in a 2‐h reaction. Inhibition experiments and theoretical modeling disclose interesting pathways for CO2 entry, formate exit, and OCN− competition, suggesting an oxidation‐state‐dependent binding mechanism of catalysis. Our results provide a different perspective for understanding the catalytic mechanism of FDH and original insights into the design of synthetic catalysts.

Microbial electrosynthesis with Clostridium ljungdahlii benefits from hydrogen electron mediation and permits a greater variety of products.

Microbial electrosynthesis (MES) is a very promising technology addressing the challenge of carbon dioxide recycling into organic compounds, which might serve as building blocks for the (bio)chemical industry. However, poor process control and understanding of fundamental aspects such as the microbial extracellular electron transfer (EET) currently limit further developments. In the model acetogen Clostridium ljungdahlii, both direct and indirect electron consumption via hydrogen have been proposed. However, without clarification neither targeted development of the microbial catalyst nor process engineering of MES are possible. In this study, cathodic hydrogen is demonstrated to be the dominating electron source for C. ljungdahlii at electroautotrophic MES allowing for superior growth and biosynthesis, compared to previously reported MES using pure cultures. Hydrogen availability distinctly controlled an either planktonic- or biofilm-dominated lifestyle of C. ljungdahlii. The most robust operation yielded higher planktonic cell densities in a hydrogen mediated process, which demonstrated the uncoupling of growth and biofilm formation. This coincided with an increase of metabolic activity, acetate titers, and production rates (up to 6.06 g L-1 at 0.11 g L-1 d-1). For the first time, MES using C. ljungdahlii was also revealed to deliver other products than acetate in significant amounts: here up to 0.39 g L-1 glycine or 0.14 g L-1 ethanolamine. Hence, a deeper comprehension of the electrophysiology of C. ljungdahlii was shown to be key for designing and improving bioprocess strategies in MES research.

Enzyme-constrained metabolic model and in silico metabolic engineering of Clostridium ljungdahlii for the development of sustainable production processes

Constraint-based genome-scale models (GEMs) of microorganisms provide a powerful tool for predicting and analyzing microbial phenotypes as well as for understanding how these are affected by genetic and environmental perturbations. Recently, MATLAB and Python-based tools have been developed to incorporate enzymatic constraints into GEMs. These constraints enhance phenotype predictions by accounting for the enzyme cost of catalyzed model´s reactions, thereby reducing the space of possible metabolic flux distributions. In this study, enzymatic constraints were added to an existing GEM of Clostridium ljungdahlii, a model acetogenic bacterium, by including its enzyme turnover numbers (kcats) and molecular masses, using the Python-based AutoPACMEN approach. When compared to the metabolic model iHN637, the enzyme cost-constrained model (ec_iHN637) obtained in our study showed an improved predictive ability of growth rate and product profile. The model ec_iHN637 was then employed to perform in silico metabolic engineering of C. ljungdahlii, by using the OptKnock computational framework to identify knockouts to enhance the production of desired fermentation products. The in silico metabolic engineering was geared towards increasing the production of fermentation products by C. ljungdahlii, with a focus on the utilization of synthesis gas and CO2. This resulted in different engineering strategies for overproduction of valuable metabolites under different feeding conditions, without redundant knockouts for different products. Importantly, the results of the in silico engineering results indicated that the mixotrophic growth of C. ljungdahlii is a promising approach to coupling improved cell growth and acetate and ethanol productivity with net CO2 fixation.

Acetate augmentation boosts the ethanol production rate and specificity by Clostridium ljungdahlii during gas fermentation with pure carbon monoxide

Recycling waste gases from industry is vital for the transition toward a circular economy. The model microbe Clostridium ljungdahlii reduces carbon from syngas and primarily produces acetate and ethanol. Here, a gas fermentation experiment is presented in chemostats with C. ljungdahlii and pure carbon monoxide (CO) as feedstock while entirely omitting yeast extract. A maximum ethanol production rate of 0.07 ± 0.01 g L−1 h−1 and a maximum average ethanol/acetate ratio of 1.41 ± 0.39 was observed under steady-state conditions. This confirmed that CO as the sole feedstock pushes the metabolism toward more reduced fermentation products. This effect was even more pronounced when 15 mM sodium acetate was added to the feed medium. An ethanol production rate of 0.23 ± 0.01 g L−1 h−1 was achieved, representing an increase of more than 240%. This increase was accompanied by an increase in cell density and selectivity toward ethanol, with a maximum average ethanol/acetate ratio of 92.96 ± 28.39. Oxygen contaminations voided this effect, although the cultures were still able to maintain a stable biomass concentration and ethanol production rate. These findings highlight the potential of CO-fermentation with acetate augmentation and the importance of preventing oxygen contaminations.

L-Cys-Assisted Conversion of H2/CO2 to Biochemicals Using Clostridium ljungdahlii.

Carbon fixation and conversion based on Clostridium ljungdahlii have great potential for the sustainable production of biochemicals (i.e., 2,3-butanediol, acetic acid, and ethanol). Here, the effects of reducing agents on the production of biochemicals from H2/CO2 using C. ljungdahlii were studied. It was found that the element S and reducing power could significantly affect the production of biochemicals, and cysteine (Cys) was better than sodium sulfide for the production of biochemicals, especially for the production of 2,3-butanediol. Moreover, comparing to the control (i.e., without the addition of Cys), the gene expression profiles indicated that the fdh and adhE1 were significantly upregulated with the addition of Cys, which involved in pathways of the CO2 fixation and ethanol production. Therefore, the irreplaceability of Cys on the production of biochemicals was both caused by its utilization as a reducing agent and its effect on the metabolic pathway. Finally, compared to the control, the production of 2,3-butanediol was increased by 2.17 times under the addition of 1.7 g/L Cys.

Boosting ethanol production rates from carbon dioxide in MES cells under optimal solventogenic conditions

Microbial Electrosynthesis (MES) has been widely applied for acetic acid (HA) production from CO2 and electricity. Ethanol (EtOH) has a higher market value than HA, and wide application in industry and as a biofuel. However, it has only been obtained sporadically and at low concentrations, probably due to sub-optimal operating conditions. This study aimed at enhancing EtOH productivity in MES cells by jointly optimising key operation parameters, including pH, H2 and CO2 partial pressure (pH2 and pCO2), and HA concentration, to promote solventogenesis. Two H-type cells were operated in fed-batch mode at −0.8 V vs. SHE with CO2 as the sole carbon source. A mixed culture, enriched with Clostridium ljungdahlii was used as the biocatalyst. The combination of low pH (<4.5) and pCO2 (<0.3 atm), along with high HA concentration (about 6 g L−1) and pH2 (>3 atm), were mandatory conditions for maintaining an efficient solventogenic culture, dominated by Clostridium sp., capable of high-rate EtOH production. The maximum EtOH production rate was 10.95 g m−2 d−1, and a concentration of 5.28 g L−1 was achieved. Up to 30 % of the electrons and 15.2 % of the carbon provided were directed towards EtOH production, and 28.1 kWh were required for the synthesis of 1 kg of EtOH from CO2. These results highlight that strict conditions are required for a continuous, reliable, EtOH production in MES cells. Future investigation should focus on improving cell configuration to achieve EtOH production at higher current densities while minimizing the electric energy input.

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.