Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)

Ellis, Lucas D.; Holwerda, Evert K.; Hogsett, David A.; Rogers, Sue; Shao, Xiongjun; Tschaplinski, Timothy J.; Thorne, Phil; Lynd, Lee R.

doi:10.1016/j.biortech.2011.09.128

Cited by 98 publications

(86 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The presence of amino acids in C. thermocellum fermentation broth has been reported before, although at lower concentrations and at lower substrate loadings [7,8,46]. Pyruvate serves as a precursor for both L-valine (4.5 g/L) and L-alanine (1.5 g/L) [8] (see Figure 4), the two amino acids produced in the highest concentrations.…”

Section: Cells Residual Substrate Nitrogen Amino Acids and Proteinsupporting

confidence: 70%

“…It is not uncommon to find pyruvate in C. thermocellum fermentations, even at lower concentrations of substrate [7,10,11]. The concentration of pyruvate found here is relatively low (325 mg/L, see Figure 3F and Figure 4).…”

Section: Overflow Metabolism Around the Pyruvate Nodementioning

confidence: 60%

“…Most of our current understanding of C. thermocellum physiology is based on fermentations with carbohydrate loadings of <10 g/L [7,[13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. There are, however, a few notable exceptions.…”

mentioning

confidence: 99%

See 2 more Smart Citations

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

Holwerda

Thorne

Olson

et al. 2014

Biotechnol Biofuels

View full text Add to dashboard Cite

Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids -predominantly L-valine and L-alanine -were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso-and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. Conclusions: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

show abstract

Section: Cells Residual Substrate Nitrogen Amino Acids and Proteinsupporting

confidence: 70%

Section: Overflow Metabolism Around the Pyruvate Nodementioning

confidence: 60%

See 1 more Smart Citation

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

Holwerda

Thorne

Olson

et al. 2014

Biotechnol Biofuels

View full text Add to dashboard Cite

show abstract

“…Their abundance in a system fed with paper as the sole exogenous substrate is surprising and suggests that extracellular proteinaceous material was abundant. Several distinct and non-exclusive protein sources can be suggested: the protein constituents of the EPS (up to 40% based on Liu and Fang, 2002); the abundant extracellular enzymes, in particular cellulosomes/cellulases or other proteins secreted by C. thermocellum (Ellis et al, 2012) and other cellulolytic strains; dead cell material present in the initial inoculum; and finally dead cell material generated during the incubation by the counter-selection of poorly competitive strains, by various stresses or by predation. Possible predators could be C. proteolyticus members, as discussed above, as well as viruses, whose presence is compatible with the four identified CRISPR-associated protein groups (Supplementary Table S15) (Bhaya et al, 2011).…”

Section: Identified Proteins Related To Central Carbon Metabolismmentioning

confidence: 99%

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

Bize²,

et al. 2013

View full text Add to dashboard Cite

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.

show abstract

“…Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3)(4)(5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6)(7)(8), as well as secreted amino acids (9,10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.…”

mentioning

confidence: 99%

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Taillefer

Rydzak

Levin

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a k cat of 45.8 s ؊1 or 14.9 s ؊1 , respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a k cat of 520.8 s ؊1 . The malic enzyme used NADP ؉ as a cofactor along with NH 4 ؉ and Mn 2؉ as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a K i of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH 4 ؉ based on the characterization of the malate dehydrogenase and malic enzyme.C lostridium thermocellum is a Gram-positive, anaerobic thermophile capable of reaching one of the highest growth rates on crystalline cellulose (1, 2). Furthermore, given its native ability to produce ethanol and H 2 , C. thermocellum is seen as an attractive microorganism for the production of biofuels via consolidated bioprocessing of lignocellulosic biomass. Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3-5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6-8), as well as secreted amino acids (9, 10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.A key pathway node involved in the interconversion of phosphoenolpyruvate (PEP) and pyruvate, which may catalyze either substrate level phosphorylation or transhydrogenation reactions between NADH to NADP ϩ , has been revisited with C. thermocellum (10, 11). The interconversion of PEP and pyruvate may be catalyzed using a number of different putative enzymes. In contrast to most clostridia and other fermentative ethanol and/or H 2 -producing organisms (7), C. thermocellum ATCC 27405 (GenBank accession number NC_009012.1) does not encode a pyruvate kinase, which catalyzes PE...

show abstract

Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)

Cited by 98 publications

References 32 publications

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Contact Info

Product

Resources

About