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ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri. In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used.IMPORTANCEThough 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.

ABSTRACT Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

ABSTRACT 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.

3-Hydroxybutyrate (3HB) is a product of interest as it is a precursor to the commercially produced bioplastic polyhydroxybutyrate. It can also serve as a platform for fine chemicals, medicines, and biofuels, making it a value-added product and feedstock. Acetogens non-photosynthetically fix CO2 into acetyl-CoA and have been previously engineered to convert acetyl-CoA into 3HB. However, as acetogen metabolism is poorly understood, those engineering efforts have had varying levels of success. 3HB, using acetyl-CoA as a precursor, can be synthesized by a variety of different pathways. Here we systematically compare various pathways to produce 3HB in acetogens and discover a native (S)-3-hydroxybutyryl-CoA dehydrogenase, hbd2, responsible for endogenous 3HB production. In conjunction with the heterologous thiolase atoB and CoA transferase ctfAB, hbd2 overexpression improves yields of 3HB on both sugar and syngas (CO/H2/CO2), outperforming the other tested pathways. These results uncovered a previously unknown 3HB production pathway, inform data from prior metabolic engineering efforts, and have implications for future physiological and biotechnological anaerobic research.

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.

ABSTRACT The 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.

Clostridium 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.

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.

Clostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L–1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L–1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.

ABSTRACT Clostridium acetobutylicum and Clostridium ljungdahlii grown in a syntrophic culture were recently shown to fuse membranes and exchange cytosolic contents, yielding hybrid cells with significant shifts in gene expression and growth phenotypes. Here, we introduce a dynamic genome-scale metabolic modeling framework to explore how cell fusion alters the growth phenotype and panel of metabolites produced by this binary community. Computational results indicate C. ljungdahlii persists in the coculture through proteome exchange during fusing events, which endow C. ljungdahlii cells with expanded substrate utilization, and access to additional reducing equivalents from C. acetobutylicum-evolved H2 and through acquisition of C. acetobutylicum-native cofactor-reducing enzymes. Simulations predict maximum theoretical ethanol and isopropanol yields that are increased by 0.64 mmol and 0.39 mmol per mmol hexose sugar consumed, respectively, during exponential growth when cell fusion is active. This modeling effort provides a mechanistic explanation for the metabolic outcome of cellular fusion and altered homeostasis achieved in this syntrophic clostridial community. IMPORTANCE Widespread cell fusion and protein exchange between microbial organisms as observed in synthetic C. acetobutylicum/C. ljungdahlii culture is a novel observation that has not been explored in silico. The mechanisms responsible for the observed cell fusion events in this culture are still unknown. In this work, we develop a modeling framework that captures the observed culture composition and metabolic phenotype, use it to offer a mechanistic explanation for how the culture achieves homeostasis, and identify C. ljungdahlii as primary beneficiary of fusion events. The implications for the events described in this study are far reaching, with potential to reshape our understanding of microbial community behavior synthetically and in nature.

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 NOX. 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 NOX 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.

ABSTRACT Microbial syntrophy is universal in nature, profoundly affecting the composition and function of microbiomes. We have recently reported data suggesting direct cell-to-cell interactions leading to electron and material exchange between the two microbes in the syntrophy between Clostridium ljungdahlii and C. acetobutylicum. Here, transmission electron microscopy and electron tomography demonstrated cell wall and membrane fusions between the two organisms, whereby C. ljungdahlii appears to invade C. acetobutylicum pole to pole. Correlative fluorescence transmission electron microscopy demonstrated large-scale exchange of proteins. Flow cytometry analysis captured the extent and dynamic persistence of these interactions. Dividing hybrid cells were identified containing stained proteins from both organisms, thus demonstrating persistence of cells with exchanged cellular components. Fluorescence microscopy and flow cytometry of one species with stained RNA and the other tagged with a fluorescent protein demonstrated extensive RNA exchange and identified hybrid cells, some of which continued to divide, while some were in an advanced C. acetobutylicum sporulation form. These data demonstrate that cell fusion enables large-scale cellular material exchange between the two organisms. Although unanticipated and never previously reported, these phenomena are likely widely distributed in nature, have profound implications for species evolution and the function of microbial communities, and could find utility in biotechnology. They may shed new light onto little-understood phenomena, such as antibiotic heteroresistance of pathogens, pathogen invasion of human tissues, and the evolutionary trajectory and persistence of unculturable bacteria. IMPORTANCE We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology.

The sustainable production of chemicals from non-petrochemical sources is one of the greatest challenges of our time. CO2 release from industrial activity is not environmentally friendly yet provides an inexpensive feedstock for chemical production. One means of addressing this problem is using acetogenic bacteria to produce chemicals from CO2, waste streams, or renewable resources. Acetogens are attractive hosts for chemical production for many reasons: they can utilize a variety of feedstocks that are renewable or currently waste streams, can capture waste carbon sources and covert them to products, and can produce a variety of chemicals with greater carbon efficiency over traditional fermentation technologies. Here we investigated the metabolism of Clostridium ljungdahlii, a model acetogen, to probe carbon and electron partitioning and understand what mechanisms drive product formation in this organism. We utilized CRISPR/Cas9 and an inducible riboswitch to target enzymes involved in fermentation product formation. We focused on the genes encoding phosphotransacetylase (pta), aldehyde ferredoxin oxidoreductases (aor1 and aor2), and bifunctional alcohol/aldehyde dehydrogenases (adhE1 and adhE2) and performed growth studies under a variety of conditions to probe the role of those enzymes in the metabolism. Finally, we demonstrated a switch from acetogenic to ethanologenic metabolism by these manipulations, providing an engineered bacterium with greater application potential in biorefinery industry.

The pH-value in fermentation broth is a critical factor for the metabolic flux and growth behavior of acetogens. A decreasing pH level throughout time due to undissociated acetic acid accumulation is anticipated under uncontrolled pH conditions such as in bottle experiments. As a result, the impact of changes in the metabolism (e.g., due to a genetic modification) might remain unclear or even unrevealed. In contrast, pH-controlled conditions can be achieved in bioreactors. Here, we present a self-built, comparatively cheap, and user-friendly multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. We tested the functionality of the MBS by cultivating the acetogen Clostridium ljungdahlii with CO2 and H2 at steady-state conditions (=chemostat). The experiments (total of 10 bioreactors) were addressing the two questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; and (2) does feeding nitrate influence the product spectrum under controlled pH conditions with CO2 and H2? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. On the one hand, our data showed high reproducibility for gas-fermentation experiments with C. ljungdahlii under standard cultivation conditions using the MBS. On the other hand, feeding nitrate as sole N-source improved growth by up to 62% and ethanol production by 2–3-fold. However, we observed differences in growth, and acetate and ethanol production rates between all nitrate bioreactors. We explained the different performances with a pH-buffering effect that resulted from the interplay between undissociated acetic acid production and ammonium production and because of stochastic inhibition events, which led to complete crashes at different operating times.

Both CO and H2 can be utilized as energy sources during the autotrophic growth of Clostridium ljungdahlii. In principle, CO is a more energetically and thermodynamically favorable energy source for gas fermentation in comparison to H2. Therefore, metabolism may vary during growth under different energy sources. In this study, C. ljungdahlii was fed with CO and/or CO2/H2 at pH 6.0 with a gas pressure of 0.1 MPa. C. ljungdahlii primarily produced acetate in the presence of H2 as an energy source, but produced alcohols with CO as an energy source under the same fermentation conditions. A key enzyme activity assay, metabolic flux analysis, and comparative transcriptomics were performed for investigating the response mechanism of C. ljungdahlii under different energy sources. A CO dehydrogenase and an aldehyde:ferredoxin oxidoreductase were found to play important roles in CO utilization and alcohol production. Based on these findings, novel metabolic schemes are proposed for C. ljungdahlii growing on CO and/or CO2/H2. These schemes indicate that more ATP is produced during CO-fermentation than during H2-fermentation, leading to increased alcohol production.

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.

The microbial production of bulk chemicals from waste gas is becoming a pertinent alternative to industrial strategies that rely on fossil fuels as substrate. Acetogens can use waste gas substrates or syngas (CO, CO2, H2) to produce chemicals, such as acetate or ethanol, but as the feed gas often contains oxygen, which inhibits acetogen growth and product formation, a cost-prohibitive chemical oxygen removal step is necessary. Here, we have developed a two-phase microbial system to facilitate acetate production using a gas mixture containing CO and O2. In the first phase the facultative anaerobic carboxydotroph Parageobacillus thermoglucosidasius was used to consume residual O2 and produce H2 and CO2, which was subsequently utilized by the acetogen Clostridium ljungdahlii for the production of acetate. From a starting amount of 3.3 mmol of CO, 0.52 mmol acetate was produced in the second phase by C. ljungdahlii. In this set-up, the yield achieved was 0.16 mol acetate/mol CO, a 63% of the theoretical maximum. This system has the potential to be developed for the production of a broad range of bulk chemicals from oxygen-containing waste gas by using P. thermoglucosidasius as an oxygen scrubbing tool.

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.

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.

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.

Cyanide is a minor constituent of crude syngas whose content depends on the feedstock and gasification procedure. It is a known poison to metal catalysts and inhibits iron-containing enzymes like carbon monoxide dehydrogenase of acetogenic organisms. Therefore, it is considered a component that has to be removed from the gas stream prior to use in chemical synthesis or syngas fermentation. We show that the growth rate and maximum biomass concentration of Clostridium ljungdahlii are unaffected by cyanide at concentrations of up to 1.0 mM with fructose as a carbon source and up to 0.1 mM with syngas as a carbon source. After the culture is adapted to cyanide it shows no growth inhibition. While the difference in growth is an increasing lag-phase with increasing cyanide concentrations, the product spectrum shifts from 97% acetic acid and 3% ethanol at 0 mM cyanide to 20% acetic acid and 80% ethanol at 1.0 mM cyanide for cultures growing on (fructose) and 80% acetic acid and 20% ethanol at 0.1 mM cyanide (syngas).

Low productivities of bioprocesses using gaseous carbon and energy sources are usually caused by the low solubility of those gases (e.g., H2 and CO). It has been suggested that increasing the partial pressure of those gases will result in higher dissolved concentrations and should, therefore, be helpful to overcome this obstacle. Investigations of the late 1980s with mixtures of hydrogen and carbon monoxide showed inhibitory effects of carbon monoxide partial pressures above 0.8 bar. Avoiding any effects of carbon monoxide, we investigate growth and product formation of Clostridium ljungdahlii at absolute process pressures of 1, 4, and 7 bar in batch stirred tank reactor cultivations with carbon dioxide and hydrogen as sole gaseous carbon and energy source. With increasing process pressure, the product spectrum shifts from mainly acetic acid and ethanol to almost only formic acid at a total system pressure of 7 bar. On the other hand, no significant changes in overall product yield can be observed. By keeping the amount of substance flow rate constant instead of the volumetric gas feed rate when increasing the process pressure, we increased the overall product yield of 7.5 times of what has been previously reported in the literature. After 90 h of cultivation at a total pressure of 7 bar a total of 4 g L−1 of products is produced consisting of 82.7 % formic acid, 15.6 % acetic acid, and 1.7 % ethanol.

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.