Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation

The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, [Y.sup.True.sub.x/ATP] = 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.sup.P-T.sub.ATP] (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-1)/n (n = mean cellodextrin DP assimilated). A mean [G.sup.P-T.sub.ATP] value of 0.52 [+ or -] 0.06 was calculated for cellulose-grown chemostat cultures, corresponding to n = 4.20 [+ or -] 0.46. Determination of intracellular [beta]-glucan radioactivity resulting from [14.sup.C]-labeled substrates showed that uptake is different for cellulose and cellobiose (G2). For [14.sup.C]-cellobiose, radioactivity was greatest for G2; substantially smaller but measurable for G1, G3, and G4; undetectable for G5 and G6; and n was [approximately equal to] 2. For [14.sup.C]-cellulose, radioactivity was greatest for G5; lower but substantial for G6, G2, and G1; very low for G3 and G4; and n was [approximately equal to] 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. cellulose hydrolysis | cellulase | cellulosome | anaerobic | thermophilic | ABC transport