Thermocellum Cultures Research Articles

Characterization of reduced carbohydrate solubilization during Clostridium thermocellum fermentation with high switchgrass concentrations

Biological conversion of lignocellulosic biomass requires high solids concentration for economical recovery of end products. Clostridium thermocellum fermentations were conducted at various concentrations of switchgrass. Significant reduction of carbohydrate solubilization has been observed for switchgrass with increasing solids concentration. Tests were conducted to understand the cause of inhibition at high solids concentration. Avicel hydrolysis by cell-free C. thermocellum cellulase or C. thermocellum fermentation on cellobiose, supplemented with supernatant or solids from C. thermocellum culture on switchgrass, revealed that inhibition was from the supernatant but not from the solids. Culture of C. thermocellum on Avicel, supplemented with supernatant from C. thermocellum monoculture or mixed consortia, indicated that Avicel solubilization was reduced by almost two-fold with the monoculture supernatant while no significant reduction was observed for the mixed consortia supernatant. Adding 8 kg/m3 commercial xylooligomers during C. thermocellum culture on Avicel reduced Avicel consumption up to 90%. Simultaneous consumption of xylooligomers was suggested for industrial application of C. thermocellum culture on high solids concentration.

Enhanced saccharification of cellulose and sugarcane bagasse by Clostridium thermocellum cultures with Triton X-100 and β-glucosidase/Cellic®CTec2 supplementation

Increased saccharification and utilization of biomass directly byC. thermocellumcultures with Triton X-100 and β-glucosidase supplementation.

CO2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2 This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.

Switchgrass (<i>Panicum virgatum</i>) Fermentation by <i>Clostridium thermocellum</i> and <i>Clostridium beijerinckii</i> Sequential Culture: Effect of Feedstock Particle Size on Gas Production

Fermentation of cellulosic biomass can be done in a single step with cellulolytic, solventogenic bacteria, such as Clostridium thermocellum. However, the suite of products is limited in consolidated bioprocessing. Fortunately, the thermophilic nature of C. thermocellum can be exploited in sequential culture. Experiments were conducted to determine the effect of feedstock particle size on fermentation by sequential cultures and to demonstrate this effect could be shown by gas production. Dual-temperature sequential cultures were conducted by first culturing with C. thermocellum (63°C, 48 h) before culturing with C. beijerinckii (35°C, 24 h). Switchgrass (2, 5 or 15 mm particle size) was the feedstock in submerged substrate (10% w/v) fermentation. The extent of fermentation was evaluated by gas production and compared by analysis of variance with Tukey’s test post hoc. C. thermocellum alone produced 78 kPa cumulative pressure (approx. 680 mL gas) when the particle size was 2 or 5 mm. The C. thermocellum cultures with 15 mm feedstock particles had a mean cumulative pressure of 15 kPa after 48 h, which was less than the 2 and 5 mm treatments (P °C) and inoculated with C. beijerinckii, and the cumulative pressures were reset to ambient, cumulative pressure values as great as 70 kPa (equivalent to an additional 670 mL gas) were produced in 24 h. Again, the longer (15 mm) particle size produced less gas (P C. beijerinckii without previous fermentation by C. thermocellum, the mean cumulative pressures were approximately 10 kPa. These results indicate that biological pretreatment with C. thermocellum increased the availability of switchgrass carbohydrates to C. beijerinckii, and that gas production is suitable method to show the effectiveness of a pretreatment.

Direct glucose production from lignocellulose using Clostridium thermocellum cultures supplemented with a thermostable β-glucosidase

BackgroundCellulases continue to be one of the major costs associated with the lignocellulose hydrolysis process. Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic bacterium that produces cellulosomes capable of efficiently degrading plant cell walls. The end-product cellobiose, however, inhibits degradation. To maximize the cellulolytic ability of C. thermocellum, it is important to eliminate this end-product inhibition.ResultsThis work describes a system for biological saccharification that leads to glucose production following hydrolysis of lignocellulosic biomass. C. thermocellum cultures supplemented with thermostable beta-glucosidases make up this system. This approach does not require any supplementation with cellulases and hemicellulases. When C. thermocellum strain S14 was cultured with a Thermoanaerobacter brockii beta-glucosidase (CglT with activity 30 U/g cellulose) in medium containing 100 g/L cellulose (617 mM initial glucose equivalents), we observed not only high degradation of cellulose, but also accumulation of 426 mM glucose in the culture broth. In contrast, cultures without CglT, or with less thermostable beta-glucosidases, did not efficiently hydrolyze cellulose and accumulated high levels of glucose. Glucose production required a cellulose load of over 10 g/L. When alkali-pretreated rice straw containing 100 g/L glucan was used as the lignocellulosic biomass, approximately 72% of the glucan was saccharified, and glucose accumulated to 446 mM in the culture broth. The hydrolysate slurry containing glucose was directly fermented to 694 mM ethanol by addition of Saccharomyces cerevisiae, giving an 85% theoretical yield without any inhibition.ConclusionsOur process is the first instance of biological saccharification with exclusive production and accumulation of glucose from lignocellulosic biomass. The key to its success was the use of C. thermocellum supplemented with a thermostable beta-glucosidase and cultured under a high cellulose load. We named this approach biological simultaneous enzyme production and saccharification (BSES). BSES may resolve a significant barrier to economical production by providing a platform for production of fermentable sugars with reduced enzyme amounts.

Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog.

BackgroundDown-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.ResultsGCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.ConclusionsDown-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

Bacterial Cellulose Hydrolysis in Anaerobic Environmental Subsystems—Clostridium thermocellumandClostridium stercorarium, Thermophilic Plant‐fiber Degraders

Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley beta-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose.

A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi.

Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolysed carob galactomannan during incubation at 60 degrees C and pH 6.5. A recombinant phage expressing mannanase activity was isolated from a library of C. thermocellum genomic DNA constructed in lambdaZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A") was hyperexpressed in Escherichia coli BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 degrees C and a pH optimum of 6.5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A" cross-reacted with a polypeptide with a molecular mass of 70000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A") bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A" consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.

Endoglucanase E, produced at high level in Escherichia coli as a lacZ′ fusion protein, is part of the Clostridium thermocellum cellulosome

The Clostridium thermocellum celE gene was expressed at high level in Escherichia coli TG1 when placed under the transcriptional and translational control of lacZ′ in pUC18; in the presence of a multicopy plasmid (pNM52) containing the lacI q gene, expression of full-length and truncated forms of celE was regulated by isopropyl-β- d-thiogalactopyranoside. Catalytically active endoglucanase E (EGE) produced by E. coli was subject to proteolytic processing. The main protein species produced from full-length and truncated forms of celE was around 40 kDa in size and had an N-terminal amino acid sequence corresponding to that derived for mature EGE from the nucleotide sequence; in addition, larger species of about 75 kDa, presumably corresponding to full-size EGE, were produced by E.coli containing the full-length celE gene. Even after removal of the signal peptide sequence, EGE produced by E. coli was secreted into the periplasm. Up to 157 bp could be deleted from the 5′ end of the celE gene without affecting the catalytic activity of EGE produced by E. coli. A polypeptide of M , 86 kDa, immunoreactive with anti-EGE antiserum, was demonstrated in the high-molecular-weight, cellulose-binding multiprotein aggregate recoverable from C. thermocellum culture supernatant.

Cloning and expression of twoClostridium thermocellum endoglucanase genes inEscherichia coli

Along with the biochemical characterization of the enzymes involved in the process of cellulose degradation byClostridium thermocellum, a genetic approach based on the cloning of the genes has been developed. From a gene bank of C.thermocellum DNA in cosmid pHC79 (1), twoE. coli clones were isolated that produced endoglucanase activity, as demonstrated by the ability of crude extracts to release reducing sugars from soluble carboxymethylcellulose (CMC) while strongly reducing the viscosity. The first clone, whose endoglucanase gene is termedcelA, produced an enzyme sharing immunological and catalytic properties with the Mr 56,000 endoglucanase (designated endoglucanase A) purified fromC. thermocellum culture supernatant by Petre et al. (2). The second clone, carrying a gene termedcelB, produced a different enzyme, which failed to cross-react immunologically with anti-endoglucanase A antiserum and had a lower activity toward trinitrophenylated CMC (1). Both genes were subcloned in vector pBR322 to yield plasmids that carryC. thermocellum DNA insertions of about 2.7 kb. The cloned sequences were used as probes to analyze restriction fragments of totalC. thermocellum DNA by Southern hybridization. Such studies demonstrated that the two genes share no homology and that they are not contiguous on theC. thermocellum chromosome. For both genes, the level of endoglucanase activity was independent of their orientation, indicating thatE. coli transcription and translation systems recognize sequences of C.thermocellum DNA and RNA that are required for proper gene expression. From the specific activity of the purified enzymes, it was calculated that each represent 0.2–0.4% of totalE. coli protein, corresponding to at least 20–40% of their expression level inC. thermocellum. Labeling of minicells containing the appropriate plasmids, followed by SDS-PAGE, demonstrated the synthesis of three immunologically related polypeptides encoded by thecelA gene, with Mr 56,000, 55,000, and 50,200, and one polypeptide of Mr 66,000 encoded by thecelB gene. In normalE. coli cells, the latter undergoes partial proteolysis and yields two active fragments of Mr 55,000 and 53,000. These fragments were purified and a specific antiserum was prepared. Ouchterlony double immunodiffusion tests and Western blot analysis showed that C.thermocellum culture supernatant contains a polypeptide of Mr 66,000 cross-reacting with antiserum made againstcelB protein synthesized inE. coli.

A modification of the Lowry method for detecting protein in media containing lignocellulosic substrates

Clostridium thermocellum culture media containing crude cellulosic substrates, prepared by the steam-explosion of wood or straw, were found to interfere strongly with the Lowry method of protein determination. Investigations into the mode of interferences demonstrated that either the intercept (blank value) and/or the slope (intensity of color development) were affected. The efficiencies of precipitation procedures using the trichloroacetic acid method and the deoxycholate-trichloroacetic acid method were compared.