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

Abstract

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.

Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites

“…It is worth noting that C. bescii cannot grow in media containing glycerol as a sole source of carbon (Hamilton‐Brehm et al., ). Increased abundance of amino acids, particularly branched chain amino acids, was also observed as products of overflow metabolism of C. thermocellum (Biswas et al., ; Holwerda et al., ) in response to genetically induced redox perturbations and increased substrate loading. Metabolic flux of the TCA cycle and associated reactions was also redistributed toward reductive reactions, resulting in increased supernatant concentrations of succinate and glutamate and decreased supernatant and intracellular concentrations of oxalomalate and 2‐oxoglutarate.…”

“…C. cellulolyticum also shunts carbon at an earlier glycolytic node (Glucose 1‐phosphate → Glucose 6‐phosphate) toward glycogen and exopolysaccharide synthesis in response to increasing substrate loads (Guedon, Desvaux, & Petitdemange, ). Pyruvate accumulation and subsequent overflow metabolism has been observed and studied in detail in C. thermocellum (Deng et al., ; Holwerda et al., ; Olson et al., ; Thompson et al., ). Accumulation of formate and hydrogen in C. thermocellum collectively restrict the reoxidation of ferredoxin and limit its availability for pyruvate:ferredoxin oxidoreductase‐enabled conversion of pyruvate to acetyl‐CoA, causing accumulation of pyruvate and pyruvate‐derived overflow metabolism products (Holwerda et al., ; Thompson & Trinh, ; Thompson et al., ).…”

“…Cell biomass was pooled from three 50 ml cultures to make one replicate for intracellular metabolite analysis, as it was needed to attain sufficient signal intensity. Cell pellets and supernatants were analyzed for intracellular and extracellular metabolites, respectively, as described previously (Holwerda et al., ).…”

CO 2 -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

“…Upon consumption of all cellobiose, cultures with added bicarbonate yielded ∼40% more cell biomass than in its absence (Table 1). Without accounting for the excreted amino acids that have been observed in overflow metabolism (3), this result increased the possibility that extra total carbon output (i.e., cell biomass, lactate) could emerge from incorporation of the inorganic carbon (bicarbonate) as suggested by the calculated apparent carbon efficiency (65.7% without bicarbonate and 75.5% with bicarbonate; Table 1). We also observed higher apparent carbon efficiency in bicarbonate-fed cultures in non-pH-controlled fermentation (Table S1) buffered with 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS).…”

“…By taking advantage of an extracellular cellulase system called the cellulosome (1), C. thermocellum can depolymerize cellulose into soluble oligosaccharides. The latter are further transported into the cells and fermented through its glycolytic pathway to pyruvate, the precursor to an array of fermentation products (e.g., H 2 , formate, lactate, acetate, ethanol, secreted amino acids) (2,3). This capability makes C. thermocellum an attractive candidate for consolidated bioprocessing of lignocellulosic biomass, a process configuration that directly converts plant biomass into biofuels and chemicals without separate additions of enzymes (4).…”