Kinetics and Metabolism of Cellulose Degradation at High Substrate Concentrations in Steady-State Continuous Cultures of
Clostridium cellulolyticum
on a Chemically Defined Medium
Authors: Desvaux Mickaël , Guedon Emmanuel , Petitdemange Henri 
Published: July 2002

ABSTRACT

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of
Clostridium cellulolyticum
with increasing amounts of carbon substrate. At a dilution rate (
D
) of 0.048 h
−1
, biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose liter
−1
the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose liter
−1
, respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of
C. cellulolyticum
metabolism in cellulose saturation was performed by varying
D
with 18.1 g of cellulose liter
−1
. Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119–130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD
+
, and
q
NADH produced
/
q
NADH used
ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as
D
rose, and (iii) the maximum growth yield ( Y

X
/
S

max

) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield ( Y

ATP

max

) (19.4 g of biomass per mol of ATP) were lowered.
C. cellulolyticum
was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327–335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells
−1
h
−1
could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells
−1
h
−1
; (ii) while the NADH/NAD
+
ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.