Search Results (12)

Clostridium ljungdahlii is a model acetogenic bacterium utilized for ethanol production from syngas, with its growth and ethanol synthesis being profoundly influenced by fermentation pH. However, the mechanistic basis of this pH-dependent regulation remains poorly understood. In this study, we systematically investigated the impact of pH on the growth and metabolic profile of C. ljungdahlii under controlled pH conditions using CO as the sole carbon and energy source. At pH 6.0, C. ljungdahlii consumed around 6.0 M carbon monoxide, producing 413 ± 43 mM acetate, 288 ± 35 mM ethanol, and 17 ± 2 mM 2,3-butanediol, with a maximum optical density (OD) of 15.9. In contrast, at pH 5.3, the strain exhibited enhanced metabolic activity, consuming around 9.6 M carbon monoxide and generating 235 ± 24 mM acetate, 756 ± 26 mM ethanol, 38 ± 4 mM 2,3-butanediol, and 28 ± 7 mM lactate, achieving a maximum OD of 30. This represents an approximate twofold increase in both ethanol production and biomass accumulation compared to pH 6.0. Proteomic and parallel reaction monitoring (PRM) analyses demonstrated that the expression levels of key enzymes in central metabolic pathways were marginally higher at pH 6.0 than at pH 5.3, indicating that the observed physiological enhancements were not attributable to differential enzyme expression but likely stemmed from variations in ATP synthesis efficiency. Further optimization experiments revealed that the optimal pH for growth and ethanol production by C. ljungdahlii under CO-sufficient and nutrient-replete conditions is approximately 5.3. These findings provide critical insights into the pH-dependent metabolic regulation of C. ljungdahlii and establish essential parameters for scaling up syngas fermentation for ethanol production. Additionally, this study offers a foundation for further exploration of the unique proton motive force-driven ATP synthesis system in C. ljungdahlii and its broader implications for metabolic network regulation. Full article

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. Full article

Utilizing syngas components CO, CO_2, and H₂ to produce fatty acids and alcohols offers a sustainable approach for biofuels and chemicals, reducing the global carbon footprint. The development of robust strains, especially for higher alcohol titers in C4 and C6 compounds, and the creation of cost-effective media are crucial. This study compared syngas fermentation capabilities of three novel strains (Clostridium carboxidivorans P20, C. ljungdahlii P14, and C. muellerianum P21) with existing strains (C. ragsdalei P11 and C. carboxidivorans P7) in three medium formulations. Fermentations in 250-mL bottles were conducted at 37 °C using H₂:CO₂:CO (30:30:40) using P11, P7, and corn steep liquor (CSL) media. Results showed that P11 and CSL media facilitated higher cell mass, alcohol titer, and gas conversion compared to the P7 medium. Strains P7, P14, and P20 formed 1.4- to 4-fold more total alcohols in the CSL medium in comparison with the P7 medium. Further, strain P21 produced more butanol (0.9 g/L) and hexanol (0.7 g/L) in the medium with CSL, offering cost advantages over P7 and P11 media containing yeast extract. Enhancing strain activity and selectivity in converting syngas into C4 and C6 alcohols requires further development, medium formulation improvements, and characterization, particularly for the new strain P21. Full article

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. Full article

In recent years, syngas fermentation has emerged as a promising means for the production of fuels and platform chemicals, with a variety of acetogens efficiently converting CO-rich gases to ethanol. However, the feasibility of syngas fermentation processes is related to the occurrence of syngas impurities such as NH₃, H₂S, and NO_X. Therefore, the effects of defined additions of NH₄⁺, H₂S, and NO₃⁻ were studied in autotrophic batch processes with C. autoethanogenum, C. ljungdahlii, and C. ragsdalei while applying continuously gassed stirred-tank bioreactors. Any initial addition of ammonium and nitrate curbed the cell growth of the Clostridia being studied and reduced the final alcohol concentrations. C. ljungdahlii showed the highest tolerance to ammonium and nitrate, whereas C. ragsdalei was even positively influenced by the presence of 0.1 g L⁻¹ H₂S. Quantitative goals for the purification of syngas were identified for each of the acetogens studied in the used experimental setup. Syngas purification should in particular focus on the NO_X impurities that caused the highest inhibiting effect and maintain the concentrations of NH₃ and H₂S within an acceptable range (e.g., NH₃ < 4560 ppm and H₂S < 108 ppm) in order to avoid inhibition through the accumulation of these impurities in the bioreactor. Full article

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. Full article

Gas fermentation by acetogens of the genus Clostridium is an attractive technology since it affords the production of biochemicals and biofuels from industrial waste gases while contributing to mitigate the carbon cycle alterations. The acetogenic model organisms C. ljungdahlii and C. autoethanogenum have already been used in large scale industrial fermentations. Among the natural products, ethanol production has already attained industrial scale. However, some acetogens are also natural producers of 2,3-butanediol (2,3-BDO), a platform chemical of relevant industrial interest. Here, we have developed a lab-scale screening campaign with the aim of enhancing 2,3-BDO production. Our study generated comparable data on growth and 2,3-BDO production of several batch gas fermentations using C. ljungdahlii and C. autoethanogenum grown on different gas substrates of primary applicative interest (CO₂ · H₂, CO · CO₂, syngas) and on different media featuring different compositions as regards trace metals, mineral elements and vitamins. CO · CO₂ fermentation was found to be preferable for the production of 2,3-BDO, and a fair comparison of the strains cultivated in comparable conditions revealed that C. ljungdahlii produced 3.43-fold higher titer of 2,3-BDO compared to C. autoethanogenum. Screening of different medium compositions revealed that mineral elements, Zinc and Iron exert a major positive influence on 2,3-BDO titer and productivity. Moreover, the CO₂ influence on CO fermentation was explored by characterizing C. ljungdahlii response with respect to different gas ratios in the CO · CO₂ gas mixtures. The screening strategies undertaken in this study led to the production of 2.03 ± 0.05 g/L of 2,3-BDO, which is unprecedented in serum bottle experiments. Full article

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. Full article

Syngas, the product of biomass gasification, can play an important role in moving towards the production of renewable chemical commodities, by using acetogenic bacteria to ferment those gaseous mixtures. Due to the complex and changing nature of biomass, the composition and the impurities present in the final biomass-derived syngas will vary. Because of this, it is important to assess the impact of these factors on the fermentation outcome, in terms of yields, productivity, and product formation and ratio. In this study, Clostridium ljungdahlii was used in a fed-batch fermentation system to analyze the effect of three different biomass-derived syngases, and to compare them to equivalent, clean syngas mixtures. Additionally, four other clean syngas mixtures were used, and the effects on product ratio, productivity, yield, and growth were documented. All biomass-derived syngases were suitable to be used as substrates, without experiencing any complete inhibitory effects. From the obtained results, it is clear that the type of syngas, biomass-derived or clean, had the greatest impact on product formation ratios, with all biomass-derived syngases producing more ethanol, albeit with lesser total productivity. Full article

The fermentation of synthesis gas, or syngas, by acetogenic bacteria can help in transitioning from a fossil-fuel-based to a renewable bioeconomy. The main fermentation products of Clostridium ljungdahlii, one of such microorganisms, are acetate and ethanol. A sensitive, robust and reproducible system was established for C. ljungdahlii syngas fermentation, and several process parameters and medium components (pH, gas flow, cysteine and yeast extract) were investigated to assess its impact on the fermentation outcomes, as well as real time gas consumption. Moreover, a closed carbon balance could be achieved with the data obtained. This system is a valuable tool to detect changes in the behavior of the culture. It can be applied for the screening of strains, gas compositions or media components, for a better understanding of the physiology and metabolic regulation of acetogenic bacteria. Here, it was shown that neither yeast extract nor cysteine was a limiting factor for cell growth since their supplementation did not have a noticeable impact on product formation or overall gas consumption. By combining the lowering of both the pH and the gas flow after 24 h, the highest ethanol to acetate ratio was achieved, but with the caveat of lower productivity. Full article

Clostridium sp. is a genus of anaerobic bacteria capable of metabolizing several substrates (monoglycerides, diglycerides, glycerol, carbon monoxide, cellulose, and more), into valuable products. Biofuels, such as ethanol and butanol, and several chemicals, such as acetone, 1,3-propanediol, and butyric acid, can be produced by these organisms through fermentation processes. Among the most well-known species, Clostridium carboxidivorans, C. ragsdalei, and C. ljungdahlii can be highlighted for their ability to use gaseous feedstocks (as syngas), obtained from the gasification or pyrolysis of waste material, to produce ethanol and butanol. C. beijerinckii is an important species for the production of isopropanol and butanol, with the advantage of using hydrolysate lignocellulosic material, which is produced in large amounts by first-generation ethanol industries. High yields of 1,3 propanediol by C. butyricum are reported with the use of another by-product from fuel industries, glycerol. In this context, several Clostridium wild species are good candidates to be used as biocatalysts in biochemical or hybrid processes. In this review, literature data showing the technical viability of these processes are presented, evidencing the opportunity to investigate them in a biorefinery context. Full article

We have established a two-stage continuous fermentation process for production of ethanol from synthesis gas (syngas) with Clostridium ljungdahlii. The system consists of a 1-L continuously stirred tank reactor as a growth stage and a 4-L bubble column equipped with a cell recycle module as an ethanol production stage. Operating conditions in both stages were optimized for the respective purpose (growth in stage one and alcohol formation in stage two). The system was fed with an artificial syngas mixture, mimicking the composition of syngas derived from lignocellulosic biomass (60% CO, 35% H₂, and 5% CO₂). Gas recycling was used to increase the contact area and retention time of gas in the liquid phase, improving mass transfer and metabolic rates. In stage two, the biocatalyst was maintained at high cell densities of up to 10 g DW/L. Ethanol was continuously produced at concentrations of up to 450 mM (2.1%) and ethanol production rates of up to 0.37 g/(L·h). Foam control was essential to maintain reactor stability. A stoichiometric evaluation of the optimized process revealed that the recovery of carbon and hydrogen from the provided carbon monoxide and hydrogen in the produced ethanol was 28% and 74%, respectively. Full article