Project description:Clostridium thermocellum is a promising CBP candidate organism capable of directly converting lignocellulosic biomass to ethanol. Low yields, productivities and growth inhibition prevent industrial deployment of this organism for commodity fuel production. Symptoms of potential redox imbalance such as incomplete substrate utilization, and fermentation products characteristic of overflow metabolism, have been observed during growth. This perceived redox imbalance may be in part responsible for the mentioned bioproductivity limitations. Toward better understanding the redox metabolism of C. thermocellum, we analyzed gene expression, using microarrays, during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which we observed to change fermentation redox potential.
Project description:Clostridium thermocellum is a promising CBP candidate organism capable of directly converting lignocellulosic biomass to ethanol. Low yields, productivities and growth inhibition prevent industrial deployment of this organism for commodity fuel production. Symptoms of potential redox imbalance such as incomplete substrate utilization, and fermentation products characteristic of overflow metabolism, have been observed during growth. This perceived redox imbalance may be in part responsible for the mentioned bioproductivity limitations. Toward better understanding the redox metabolism of C. thermocellum, we analyzed gene expression, using microarrays, during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which we observed to change fermentation redox potential. High quality RNA was extracted from C. thermocellum grown on cellobiose in chemostat culture and exposed, separately, to methyl viologen and hydrogen peroxide. Transcriptome profiles were obtained at seven time points during actively growing fermentations, 3 minutes, 15 minutes, 35 minutes, 7 hours, 14 hours, 50 hours, and 60 hours after beginning exposure to each stressor. Exposure treatments were carried out in duplicate and reference/untreated samples were taken before and between treatments, after flushing of stressor chemicals and re-equilibration of growth conditions.
Project description:A region of genomic DNA from Clostridium kluyveri was cloned in Escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene. The gene region (6,575 bp) contained several open reading frames which encoded the coenzyme A (CoA)- and NADP+-dependent succinate-semialdehyde dehydrogenase (sucD), the 4-hydroxybutyrate dehydrogenase (4hbD), and a succinyl-CoA;CoA transferase (cat1), as analyzed by heterologous expression in E. coli. An open reading frame encoding a putative membrane protein (orfY) and the 5' region of a gene encoding a sigma 54-homologous sigma factor (sigL) were identified as well. Transcription was investigated by Northern (RNA) blot analysis. Protein sequence comparisons of SucD and 4HbD revealed similarities to the adhE (aad) gene products from E. coli and Clostridium acetobutylicum and to enzymes of the novel class (III) of alcohol dehydrogenases. A comparison of CoA-dependent aldehyde dehydrogenases is presented.
Project description:Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions.
Project description:BackgroundThe product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures.ResultsHere, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n-caproic acid, which were up to 40 mM day-1 (4.64 g L-1 day-1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n-caproic acid production. In addition, n-caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions.ConclusionsClostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.