Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation

Abstract

The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated for cellulose-grown chemostat cultures, corresponding to n = 4.20 +/- 0.46. Determination of intracellular beta-glucan radioactivity resulting from 14C-labeled substrates showed that uptake is different for cellulose and cellobiose (G2). For 14C-cellobiose, radioactivity was greatest for G2; substantially smaller but measurable for G1, G3, and G4; undetectable for G5 and G6; and n was approximately 2. For 14C-cellulose, radioactivity was greatest for G5; lower but substantial for G6, G2, and G1; very low for G3 and G4; and n was approximately 4. These results indicate that: (i) C. thermocellum hydrolyzes cellulose by a different mode of action from the classical mechanism involving solubilization by cellobiohydrolase; (ii) bioenergetic benefits specific to growth on cellulose are realized, resulting from the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic cleavage of beta-glucosidic bonds; and (iii) these benefits exceed the bioenergetic cost of cellulase synthesis, supporting the feasibility of anaerobic biotechnological processing of cellulosic biomass without added saccharolytic enzymes.

Fig. 1.

Plot for determination of and m based on Eq. 2. Data are calculated from steady-state continuous cultures of C. thermocellum growing on cellobiose with a feed concentration of ≈5 g/liter (see Table 3 which is published as supporting information on the PNAS web site, for details).

Fig. 2.

Predicted (○) and measured (•) values as a function of n. Experimental points are calculated from steady-state continuous cultures of C. thermocellum growing on cellodextrins of length 2-6 as well as cellulose (Avicel). The dashed line denotes the mean value of for cellulose fermentation, with the shaded region denoting the standard deviation for independent steady-state fermentation runs carried out at various dilution rates (see Tables 4 and 5, which are published as supporting information on the PNAS web site, for details).

Fig. 3.

Radioactivity of intracellular cellodextrins following addition of [¹⁴C]cellobiose (0.05 μCi/ml broth, (A) and phosphoric acid-swollen [¹⁴C]cellulose (0.5 μCi/ml broth, B). ▴, glucose; •, cellobiose; ▪, cellotriose; ▾, cellotetraose; •, cellopentaose; ▪, cellohexaose; and ♦, number average degree of polymerization of intracellular cellodextrin.

Fig. 4.

ATP generation (A) and demand (B) for C. thermocellum growing on cellulose (Avicel). Data are calculated from steady-state continuous cultures of C. thermocellum growing on Avicel (see Table 5 for details).