Biofuel and chemical production from carbon one industry flux gas by acetogenic bacteria.

Carbon one industry flux gas generated from fossil fuels, various industrial and domestic waste, as well as lignocellulosic biomass provides an innovative raw material to lead the sustainable development. Through the chemical and biological processing, the gas mixture composed of CO, CO2, and H2, also termed as syngas, is converted to biofuels and high-value chemicals. Here, the syngas fermentation process is elaborated to provide an overview. Sources of syngas are summarized and the influences of impurities on biological fermentation are exhibited. Acetogens and carboxydotrophs are the two main clusters of syngas utilizing microorganisms, their essential characters are presented, especially the energy metabolic scheme with CO, CO2, and H2. Synthetic biology techniques and microcompartment regulation are further discussed and proposed to create a high-efficiency cell factory. Moreover, the influencing factors in fermentation and products in carboxylic acids, alcohols, and others such like polyhydroxyalkanoate and poly-3-hydroxybutyrate are addressed. Biological fermentation from carbon one industry flux gas is a promising alternative, the latest scientific advances are expatiated hoping to inspire more creative transformation.

Functional and structural analysis of catabolite control protein C that responds to citrate.

Catabolite control protein C (CcpC) belongs to the LysR-type transcriptional regulator (LTTR) family, which regulates the transcription of genes encoding the tricarboxylic acid branch enzymes of the TCA cycle by responding to a pathway-specific metabolite, citrate. The biological function of CcpC has been characterized several times, but the structural basis for the molecular function of CcpC remains elusive. Here, we report the characterization of a full-length CcpC from Bacillus amyloliquefaciens (BaCcpC-FL) and a crystal structure of the C-terminal inducer-binding domain (IBD) complexed with citrate. BaCcpC required both dyad symmetric regions I and II to recognize the citB promoter, and the presence of citrate reduced citB promoter binding. The crystal structure of CcpC-IBD shows two subdomains, IBD-I and IBD-II, and a citrate molecule buried between them. Ile100, two arginines (Arg147 and Arg260), and three serines (Ser129, Ser189, and Ser191) exhibit strong hydrogen-bond interactions with citrate molecules. A structural comparison of BaCcpC-IBD with its homologues showed that they share the same tail-to-tail dimer alignment, but the dimeric interface and the rotation between these molecules exhibit significant differences. Taken together, our results provide a framework for understanding the mechanism underlying the functional divergence of the CcpC protein.

Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide.

Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD+ during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (adhE) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1 MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in Clostridium ljungdahlii A metabolic experiment using 13C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases.IMPORTANCE Ethanol production from carbon monoxide (CO) as a carbon and energy source by Clostridium ljungdahlii and "Clostridium autoethanogenum" is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of C. ljungdahlii in the future.

Correction to: Emerging technologies for the pretreatment of lignocellulosic materials for bio-based products.

This corrects the article "Emerging technologies for the pretreatment of lignocellulosic materials for bio-based products" in volume 104, with page no 455-473, (https://doi.org/10.1007/s00253-019-10158-w).

Energy Conservation and Carbon Flux Distribution During Fermentation of CO or H2/CO2 by Clostridium ljungdahlii.

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.

Emerging technologies for the pretreatment of lignocellulosic materials for bio-based products.

Exploring a cheap and clean renewable energy has become a common destination round the world with the depletion of oil resources and the concerns of increasing energy demands. Lignocellulosic biomass is the most abundant renewable resource in the biosphere, and the total biomass formed by plant photosynthesis reached more than 200 billion tons every year. Cellulase and hemicellulose and lignin degradation enzymes, the efficient biocatalyst, could efficiently convert the lignocellulosic biomass into sugars that could be further processed into biofuels, biochemical, and biomaterial for human requirement. The utilization and conversion of cellulosic biomass has great significance to solve the problems such as environmental pollution and energy crisis. Lignocellulosic materials are widely considered as important sources to produce sugar streams that can be fermented into ethanol and other organic chemicals. Pretreatment is a necessary step to overcome its intrinsic recalcitrant nature prior to the production of important biomaterial that has been investigated for nearly 200 years. Emerging research has focused in order of economical, eco-friendly, and time-effective solutions, for large-scale operational approach. These new mentioned technologies are promising for lignocellulosic biomass degradation in a huge scale biorefinery. This review article has briefly explained the emerging technologies especially the consolidated bioprocessing, chemistry, and physical base pretreatment and their importance in the valorization of lignocellulosic biomass conversion.

Modulation of the Acetone/Butanol Ratio during Fermentation of Corn Stover-Derived Hydrolysate by Clostridium beijerinckii Strain NCIMB 8052.

Producing biobutanol from lignocellulosic biomass has shown promise to ultimately reduce greenhouse gases and alleviate the global energy crisis. However, because of the recalcitrance of a lignocellulosic biomass, a pretreatment of the substrate is needed which in many cases releases soluble lignin compounds (SLCs), which inhibit growth of butanol-producing clostridia. In this study, we found that SLCs changed the acetone/butanol ratio (A/B ratio) during butanol fermentation. The typical A/B molar ratio during Clostridium beijerinckii NCIMB 8052 batch fermentation with glucose as the carbon source is about 0.5. In the present study, the A/B molar ratio during batch fermentation with a lignocellulosic hydrolysate as the carbon source was 0.95 at the end of fermentation. Structural and redox potential changes of the SLCs were characterized before and after fermentation by using gas chromatography/mass spectrometry and electrochemical analyses, which indicated that some exogenous SLCs were involved in distributing electron flow to C. beijerinckii, leading to modulation of the redox balance. This was further demonstrated by the NADH/NAD+ ratio and trxB gene expression profile assays at the onset of solventogenic growth. As a result, the A/B ratio of end products changed significantly during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source compared to glucose as the carbon source. These results revealed that SLCs not only inhibited cell growth but also modulated the A/B ratio during C. beijerinckii butanol fermentation.IMPORTANCE Bioconversion of lignocellulosic feedstocks to butanol involves pretreatment, during which hundreds of soluble lignin compounds (SLCs) form. Most of these SLCs inhibit growth of solvent-producing clostridia. However, the mechanism by which these compounds modulate electron flow in clostridia remains elusive. In this study, the results revealed that SLCs changed redox balance by producing oxidative stress and modulating electron flow as electron donors. Production of H2 and acetone was stimulated, while butanol production remained unchanged, which led to a high A/B ratio during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source. These observations provide insight into utilizing C. beijerinckii to produce butanol from a lignocellulosic biomass.

Spontaneous large-scale autolysis in Clostridium acetobutylicum contributes to generation of more spores.

Autolysis is a widespread phenomenon in bacteria. In batch fermentation of Clostridium acetobutylicum ATCC 824, there is a spontaneous large-scale autolysis phenomenon with significant decrease of cell density immediately after exponential phase. To unravel the role of autolysis, an autolysin-coding gene, CA_C0554, was disrupted by using ClosTron system to obtain the mutant C. acetobutylicum lyc::int(72). The lower final cell density and faster cell density decrease rate of C. acetobutylicum ATCC 824 than those of C. acetobutylicum lyc::int(72) indicates that CA_C0554 was an important but not the sole autolysin-coding gene responding for the large-scale autolysis. Similar glucose utilization and solvents production but obvious lower cell density of C. acetobutylicum ATCC 824 comparing to C. acetobutylicum lyc::int(72) suggests that lysed C. acetobutylicum ATCC 824 cells were metabolic inactive. On the contrary, the spore density of C. acetobutylicum ATCC 824 is 26.1% higher than that of C. acetobutylicum lyc::int(72) in the final culture broth of batch fermentation. We speculated that spontaneous autolysis of metabolic-inactive cells provided nutrients for the sporulating cells. The present study suggests that one important biological role of spontaneous large-scale autolysis in C. acetobutylicum ATCC 824 batch fermentation is contributing to generation of more spores during sporulation.

Characterization of an acetoin reductase/2,3-butanediol dehydrogenase from Clostridium ljungdahlii DSM 13528.

Acetoin reductase catalyzes the formation of 2,3-butanediol from acetoin. In Clostridium ljungdahlii DSM 13528, the gene CLJU_c23220 encoding the putative Zn(2+)-dependent alcohol dehydrogenase was cloned and expressed in Escherichia coli. The recombinant enzyme, CLAR, can catalyze the conversion of acetoin to 2,3-butanediol with NADPH as the cofactor. Furthermore, the gene CLJU_c23220 was introduced into Clostridium acetobutylicum ATCC 824 and the transformant was conferred the capacity of 2,3-butanediol production. In batch fermentation the transformant produced up to 3.1g/L of 2,3-butanediol, as well as acetone, butanol and ethanol (ABE, 17.8 g/L) in amounts similar to those produced by the wild type strain. This study provides conclusive evidence at the protein level that CLJU_c23220 is the key gene responsible for the conversion of acetoin to 2,3-butanediol in C. ljungdahlii DSM 13528. Moreover, the C. acetobutylicum ATCC 824 was modified via one-step metabolic engineering to produce 2,3-butanediol without influencing the ABE production.

Comparative transcriptome analysis between csrA-disruption Clostridium acetobutylicum and its parent strain.

The genome of Clostridium acetobutylicum contains the gene encoding CsrA, a carbon storage regulator. We investigated the function of CsrA in C. acetobutylicum by insertionally inactivating the encoding gene, CA_C2209 using the ClosTron. Disruption of csrA obviously decreases the growth of the organism and reduces the yield of acetone, butanol and ethanol (ABEs). Like the csrA in Escherichia coli, RNA-seq and ß-galactosidase analysis revealed that csrA in C. acetobutylicum was closely involved in regulating multiple pathways including flagella assembly, oligopeptide transporting, iron uptake, and central carbon metabolism. It has also been newly demonstrated that csrA in C. acetobutylicum is related to the regulation of pathways involved in the phosphotransferase transporting systems, synthesis of riboflavin, and stage III sporulation. This research represented the first investigation of global regulation by CsrA in the strain belonging to Gram-positive bacteria through transcriptome analysis and provided the important theoretical evidence for improving solvent production by transcriptor engineering in C. acetobutylicum.

Physiological response of Clostridium ljungdahlii DSM 13528 of ethanol production under different fermentation conditions.

In this study, cell growth, gene expression and ethanol production were monitored under different fermentation conditions. Like its heterotrophical ABE-producing relatives, a switch from acidogenesis to solventogenesis of Clostridium ljungdahlii during the autotrophic fermentation with CO/CO2 could be observed, which occurred surprisingly in the late-log phase rather than in the transition phase. The gene expression profiles indicated that aor1, one of the putative aldehyde oxidoreductases in its genome played a critical role in the formation of ethanol, and its transcription could be induced by external acids. Moreover, a low amount of CaCO3 was proved to have positive influences on the cell density and substrate utilization, followed by an increase of over 40% ethanol and 30% acetate formation.