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Nitrate Feed Improves Growth and Ethanol Production of Clostridium ljungdahlii With CO2 and H2, but Results in Stochastic Inhibition Events.

ABSTRACT: 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.

SUBMITTER: Klask CM

PROVIDER: S-EPMC7219301 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Nitrate Feed Improves Growth and Ethanol Production of Clostridium ljungdahlii With CO2 and H2, but Results in Stochastic Inhibition Events.

Klask Christian-Marco CM Kliem-Kuster Nicolai N Molitor Bastian B Angenent Largus T LT

Frontiers in microbiology 20200506

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 sel ...[more]

PMID: 32435236

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Project description:UnlabelledMost acetogens can reduce CO2 with H2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO2 and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD(+) oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2 and H2.ImportanceEthanol formation from syngas (H2, CO, and CO2) and from H2 and CO2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2 and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

Project description:Clostridium ljungdahlii derives energy by acetogenesis as a lithotrophic pathway and as part of various organotrophic pathways. Its recently sequenced genome has made it possible to discover changes in gene expression that occur in different growth modes, from which strategies for biofuel production may be developed. C. ljungdahlii was grown with fructose as an organoheterotrophic substrate and also lithoautotrophically, either on syngas or with H2 as the electron donor and CO2 as the electron acceptor. RNA extracted from all three conditions was analyzed by hybridization to a microarray, and gene expression was compared quantitatively by RNA-Seq of C. ljungdahlii grown with fructose and with H2 and CO2. Results. Strongly upregulated (> 10-fold, p < 0.05) genes with both syngas and H2/CO2 encode enzymes that degrade aspartate and arginine through the urea cycle to control the release of ammonia from amino acids. Numerous genes for uptake and degradation of peptides and amino acids, response to sulfur starvation, and molybdopterin-dependent pathways were also significantly (> 2-fold, p < 0.05) upregulated, along with three potentially NADPH-producing pathways for which the key metabolites are (S)-malate, ornithine and 6-phospho-D-gluconate. Upregulation of genes implicated in quorum sensing, sporulation and cell wall remodeling suggests a global and multicellular response to lithoautotrophic conditions. With syngas, (R)-lactate dehydrogenase and associated electron transfer flavoproteins were upregulated, representing a route of electron transfer from ferredoxin to NAD that is independent of the proton-translocating Rnf complex. With H2/CO2, a flavodoxin and histidine biosynthesis enzymes were upregulated. Genes for degradation of purine bases entering the cell by facilitated diffusion were upregulated in lithotrophic cells, whereas genes for degradation of adenine or adenosine derivatives entering by active transport were significantly (2-fold, p < 0.05) downregulated. The most downregulated genes, after those specific to fructose metabolism, encode enzymes of pyrimidine and purine biosynthesis, arginine fermentation to ornithine, threonine biosynthesis and phosphate uptake. Genes for biogenesis of an intracytoplasmic microcompartment were downregulated, within which (S)-1,2-propanediol dehydratase and other enzymes may dispose of methylglyoxal, a toxic byproduct of glycolysis, as 1-propanol. Several redox-active proteins, both cytoplasmic and membrane-associated, and predicted cell surface proteins were identified as differentially regulated. Conclusion. The transcriptomic profiles of C. ljungdahlii in lithoautotrophic and organoheterotrophic growth modes indicate large-scale physiological and metabolic differences, observations that may guide the production of biofuels and commodity chemicals with this species.

Dataset Information

Nitrate Feed Improves Growth and Ethanol Production of Clostridium ljungdahlii With CO2 and H2, but Results in Stochastic Inhibition Events.

Publications

Nitrate Feed Improves Growth and Ethanol Production of <i>Clostridium ljungdahlii</i> With CO<sub>2</sub> and H<sub>2</sub>, but Results in Stochastic Inhibition Events.

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