Figure 3: In vitro and in vivo evidence for extracellular cellulose digestion and cellobiose uptake.

Error bars (b,e) represent s.d. derived from four biological replicates each including two technical replicates. (a) In vitro digestion of carboxymethyl cellulose (CMC), filter paper and Avicel (Avi) using culture supernatants of Chlamydomonas grown with CMC or without supplements (MM). Chlorella was grown in the presence of CMC and served as a control. Thin-layer chromatography was used to identify hydrolysis products after the indicated incubation times (h). Glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4) and cellopentaose (G5) served as standards. (b) Detection and quantification of CB in culture supernatants of C. reinhardtii cells grown in MM supplemented with crystalline cellulose (FP) by GC/MS. CB (mg CB × g⁻¹ FP) in the culture supernantant (y axis) was monitored during cultivation (x axis; days (d) of cultivation). Error bars represent s.d. (c) Detection of CB in the intracellular metabolome of C. reinhardtii cells grown in 0.1% (w/v) FP-containing MM (MM+FP). Gas chromatographic (GC) profiles (i, ii) and mass spectrometric (MS) analysis (iii, iv) of the eluate collected at a retention time of 41.96 min (black rectangle: i, ii). The peak at 361.13 m/z (black rectangle, iii) represents the dominant mass of CB. Cell samples obtained by cultivation in FP-free media were used as a control (MM). (d) Import and assimilation of CB demonstrated by incubation of C. reinhardtii in the presence (³H-CB) or absence (control) of tritium-labelled CB. An autoradiogram of SDS–PAGE fractionated and blotted proteins (ARG) is shown together with a Coomassie (CBS) stain. (e) Growth analysis of a non-phototrophic mutant (cc4148 (FUD16) cultivated in the presence (blue curve) or absence (black curve, MM) of 3 mM CB (MM+CB). Cell densities (y axis) are given along with the time of cultivation (x axis) in days (d).