C. reinhardtii secretes functional endo-β-1,4-glucanases

In a first step we tested the ability of C. reinhardtii to degrade the water-soluble cellulose derivative carboxymethyl cellulose (CMC). Active digestion of CMC (Fig. 1a) by two different C. reinhardtii wild-type strains (cc124, cw10) within Congo red plate assays22 caused a halo formation, suggesting CMCase secretion into the solid medium. In contrast, this phenotype could not be observed for the closely related microalga Chlorella kessleri, which uses a cellulosic cell wall23. Zymographic CMCase detection in culture supernatant samples (Fig. 1b) demonstrated a constant increase in the CMCase activity only during cultivation in the presence of CMC. This activity was exclusively found in the culture supernatant (Fig. 1c), and identification of two highly similar (64% identity) CMCases CrCel9B (UniProtKB A8JFG8) and CrCel9C (UniProtKB A8JFH1) within the secretome of this alga was achieved by affinity-based enrichment on microcrystalline cellulose (Avicel) in conjunction with mass spectrometric analysis (Fig. 1d and Supplementary Table S3). Both proteins belong to GH family 9 (GHF9) with high homology to endo-β-1,4-glucanases from lower (for example, UniProtKB Q64I76) and higher (UniProtKB O77045) termites, as well as from earthworms (UniProtKB B9A7E3)24. Homology-based modelling25 identified the GHF9 endo/exocellulase CelE4 from T. fusca26 as a structural homologue of CrCel9B and endoglucanase AaCel9A from A. acidocaldarius27 as a homologue of CrCel9C (Supplementary Fig. S1 and Supplementary Table S1). In contrast to endogenous termite endoglucanases28, which typically contain only a catalytic GHF9 domain (Supplementary Fig. S1, NtEG) the algal enzymes possess an additional unknown type of module at either the carboxy (CrCel9B) or amino terminus (CrCel9C). Enrichment of the C. reinhardtii cellulases by purification on Avicel argues for a cellulose-binding function of the extra modules in CrCel9B/C, as efficient binding of GHF9 endoglucanases to microcrystalline cellulose requires binding modules in addition to the catalytic domain29.

Figure 1: C. reinhardtii cells secrete cellulolytic enzymes after media supplementation with carboxymethyl cellulose. (a) Congo red staining (CRS) of minimal media (MM) agar plates with C. reinhardtii (cc124; cw10) and Chlorella kessleri wild-type colonies supplemented with 0.1% (w/v) carboxymethyl cellulose (CMC), 5 days after inoculation and growth under continuous illumination with 40 μmol m−2 s−1. (b) Cellulolytic activity of C. reinhardtii cc124 wild-type cultures cultivated in MM with (+) or without (−) 0.1% (w/v) CMC, visualized by zymogram assays of the culture supernatant taken at the indicated time points (d, days of cultivation) of cultivation. (c) Localization of the CMCase activity by zymography (CRS) using subcellular fractions of C. reinhardtii cells collected after 8 days of cultivation in CMC-containing MM (+) or CMC-free MM (−) and colloidal Coomassie staining (CCS) served as a loading control. (d) Identification of two secreted C. reinhardtii CMCases by tandem mass spectrometry (MS/MS) de novo sequencing after Avicel purification from a secretome sample (input) and zymographic (CRS) detection. Full size image

Chlamydomonas cells utilize exogenous cellulosic material

Secretion of active endoglucanases degrading water-soluble cellulose (Fig. 1) indicated the existence of an enzymatic system for extracellular cellulose digestion in C. reinhardtii. Consequently, we analysed whether degradation products are used to fuel growth, which would imply the utilization of cellulosic material. Extracellular cellulose utilization by C. reinhardtii was demonstrated by growth experiments using high-salt minimal medium (MM) and air levels of carbon dioxide (Fig. 2a). Addition of CMC (MM+CMC) or filter paper (MM+FP) to Chlamydomonas wild-type cultures caused an increased exponential growth rate and a higher final cell density (Fig. 2a). Within this photoheterotrophic growth regime, it is not possible to discriminate between the contributions of photosynthesis and heterotrophic cellulose assimilation. We therefore analysed the growth-enhancing effect of cellulosic material in cultures of photosynthetic mutants from C. reinhardtii (Fig. 2b), which were supplemented with only a very low concentration of acetate (10% of the standard TAP (tris-acetate phosphate) medium30 content), required for initiation of cell growth before an induction of cellulase expression. When growth of the cc4148 (FUD16) non-photoautotrophic mutant31 in cellulose-containing and cellulose-free medium was compared, a biphasic curve resulted (Fig. 2b). Acetate assimilation in the initial phase (Fig. 2b, right y axis, dotted lines, first 2 days) led to growth with comparable rates for all cultures, but an earlier stagnation of growth in the cellulose-free culture (Fig. 2b, left y axis, black continuous curve) indicated assimilation of cellulose in the FP/CMC-containing cultures (Fig. 2b, red and green continuous curves) after the medium was depleted of acetate (Fig. 2b, right y axis, dotted lines, day 0 versus days 2–12), which takes only 2–3 days even if the standard acetate amount is used for cultivation32. It is important to note that all cultures grew at almost identical rates under acetate-replete conditions, so that differences in the assimilation rate of acetate can be excluded as the cause for the higher growth rate in the cellulose-containing cultures. Addition of cellulosic material to medium containing a lowered amount of acetate caused a delayed transition from the exponential to the stationary growth phase and increased the final cell density considerably (249±17% FP and 295±15% CMC versus 100% cellulose-free culture, values±(s.d.) with n=9). Similar results (Fig. 2c, 156±5.85% (s.d./n=6) with FP versus 100% without cellulose on day 6) were obtained with another photosynthetic mutant cc4147 (FUD7)33. In addition to experiments carried out in MM containing a low amount of acetate, cellulose assimilation of photosynthetic mutant FUD16 was also analysed under conditions where cellulose was the sole organic carbon source present in the medium (Fig. 2d). Under these conditions, the photosynthetic mutant has to establish a cellulose-degrading environment without the aid of low acetate amounts in the initial phase of the growth experiment, which causes an extended lag-phase (Fig. 2d, green curve, first 4 days). However, addition of cellulose (Fig. 2d, MM+FP, green curve) resulted in growth of photosynthetic mutant FUD16 and caused a significantly higher final cell density (153±7.3% (s.d./n=6) FP versus 100% MM). Chlamydomonas reinhardtii is therefore capable of heterotrophic cellulose assimilation.

Figure 2: Improved growth phenotype of C. reinhardtii cell cultures after addition of cellulosic material. Error bars represent s.d. derived from three biological replicates, each including at least two technical replicates. (a–c) Growth analyses of (a) wild-type cc124, (b) the non-phototrophic mutant cc4148 (FUD16) and (c) the non-phototrophic mutant cc4147 (FUD7), grown in MM without or with the addition of CMC (MM+CMC) or FP (MM+FP). For cultivation in b and c, MM were supplemented with 0.1 g l−1 acetate. Acetate concentration (dotted lines) in the medium was monitored and plotted on the right y axis. (d) Mean cell densities of the non-phototrophic mutant FUD16 cultivated in MM or in MM supplemented with FP (MM+FP). (e) Increases in final cell density (left y axis, black bars) and specific growth rate (right y axis, white bars) caused by cellulose addition (CMC; +) to C. reinhardtii or Chlorella kessleri cultures. The growth-enhancing effect was determined in the presence of acetate or various carbon dioxide concentrations (high: 5% (v/v)/enriched air; low: ~0.04% (v/v)/air bubbling; very low: no bubbling) in MM. Final cell densities are given as relative changes (CMC-free cultivation set to 100%). (f) Final cell density (left y axis, black bars) and specific growth rate differences (right y axis, white bars) of air-bubbled cultures caused by addition of CB (MM+CB), Avicel (MM+Avi), FP (MM+FP) or CMC in the presence of antibiotic and fungicide (MM+CMC*). MM cultivation was set to 100%. Full size image

More detailed analyses of the growth-enhancing effect observed after addition of CMC (Fig. 2a) revealed that the growth-stimulating effect of CMC (Fig. 2e, (+)) in comparison with CMC-free MM (Fig. 2e, (−)) was inversely correlated to the availability of other carbon sources, such as carbon dioxide (high, low, very low CO 2 ) or acetate, indicating a special relevance of cellulose as an alternative carbon source (Fig. 2e, right y axis, white bars). Under very low CO 2 conditions, where Chlamydomonas showed the largest specific growth rate increase caused by CMC addition, the growth rate of Chlorella kessleri was unaffected (Fig. 2e, Chlorella, (−) versus (+)), which is explained by the fact that Chlorella does not secrete CMCases (Fig. 1a). The largest final cell density difference (209±11.9% (s.d.) with CMC versus 100% without CMC) between CMC-containing and CMC-free cultures was observed, when liquid cultures were bubbled with air, so that this condition was chosen for further analyses (Fig. 2e, low CO 2 , left y axis, black bars). Importantly, addition of an antibiotic (25 μg ml−1 kasugamycin34) together with a fungicide (5 μg ml−1 carbendacime35) demonstrated that the growth-enhancing effect of CMC is not caused by a contaminant-assisted digestion process (Fig. 2f, MM+CMC*). Growth rate and final cell density were even more increased when FP as a crystalline cellulose substrate was added (Fig. 2f, MM+FP) and even the addition of Avicel as a very recalcitrant substrate improved growth, although to a smaller extent (Fig. 2f, MM+Avi). Cellobiose, which is the final product of extracellular cellulose hydrolysis for many cellulolytic organisms, increased the final cell density of C. reinhardtii cultures when it was added as a culture supplement (Fig. 2f, MM+CB). In conclusion, the addition of cellulosic compounds or of cellulose breakdown products, such as CB, clearly enhances the growth rate of this microalga.

C. reinhardtii internalizes cellulose breakdown products

Utilization of cellulose for cell growth requires enzymatic degradation to internalizable fragments (cellodextrins, CB). We therefore determined the specific cellulolytic activity found in crude C. reinhardtii supernatants on crystalline (FP, Avicel) and non-crystalline (CMC) substrates (Table 1). In contrast to Chlorella, where no activity could be detected on either substrate, Chlamydomonas displayed activity on all analysed substrates with the highest activity on soluble cellulose (Table 1, CMC) and comparable activities for both crystalline substrates (Table 1, Avicel and FP). Not surprisingly, specific activities measured for C. reinhardtii were substantially lower than the activities previously reported for cellulolytic bacteria and fungi (87- to 218-fold CMC; 19- to 77-fold Avicel; 9- to 61-fold FP)36,37,38,39 underscoring that this photosynthetic organism uses cellulose digestion as an extra mode of organic carbon acquisition and not as a main heterotrophic growth strategy, which would apply to saprophytes.

Table 1 Hydrolytic activity of Chlamydomonas supernatant proteins on cellulosic substrates. Full size table

In vitro hydrolysis of CMC using C. reinhardtii culture supernatants and subsequent analysis of digestion products revealed the formation of cellodextrins (cellotriose, cellotetraose and cellopentaose) as well as CB, only after cultivation in the presence of CMC (Fig. 3a, CMC versus MM), and again no CMCase activity could be detected for Chlorella kessleri (Fig. 3a, Chlorella). As suggested by the increased growth rate (Fig. 2f) using FP and Avicel, even these crystalline substrates could be digested to CB (Fig. 3a, FP and Avi).

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). Full size image

We next intended to demonstrate the conversion of crystalline cellulose (FP) to CB in situ. The supernatant of a culture growing in MM and supplemented with FP was subjected to gas chromatography/mass spectrometry (GC/MS) measurements (Fig. 3b) to detect and quantify CB. In good correlation with our growth analyses (Fig. 2a), which showed significant changes in the growth rate between FP-containing and cellulose-free cultures from day 9 onwards, CB could be detected on day 9 after the start of cultivation, and increased further (Fig. 3b, grey bars, 5.3±0.9 mg CB per g of added FP on day 13). Apart from detection in the supernatant, CB could also be found inside the cells (Fig. 3c, 0.4±0.07 mg CB per g of dried biomass).

The detection of CB among the in vitro and in situ hydrolysis products, together with the finding that CB addition to MM improved the growth of C. reinhardtii (Fig. 2f, MM+CB), suggested that CB is a cellulose breakdown product, which can be internalized by C. reinhardtii (Fig. 3c). Consequently, CB transport into C. reinhardtii cells and the ability to assimilate this disaccharide were examined. Addition of radiolabelled 3H-CB to C. reinhardtii cultures resulted in the incorporation of radiolabel into proteins, demonstrating active uptake and further utilization of external CB (Fig. 3d, ARG, 3H-CB). The ability of C. reinhardtii to grow strictly heterotrophic on CB was further proven by growth analyses (Fig. 3e) using the non-phototrophic mutant cc4148 (FUD16)31, which were conducted in the same way as FP and CMC growth-enhancing effects were determined (Fig. 2b), except for the replacement of cellulose by CB. Again, a biphasic curve was observed with almost identical growth rates of CB-containing and CB-free cultures under acetate-replete conditions (until day 2 after the start of cultivation). After the second day of cultivation, the CB-containing culture showed an increased growth rate (Fig. 3e, blue curve, MM+CB), resulting in a higher final cell density (220±6.7% for MM+CB versus MM set to 100% on day 10). The demonstrated ability to import CB might explain, why this green microalga does not possess a hexose uptake system17,18. So far, only a few CB transporters have been identified belonging to the major facilitator superfamily40 or the ATP-binding cassette transporter family41, and members of both are encoded by the Chlamydomonas nuclear genome (for example, UniProtKB A8J114/A8ILE4).

In vitro hydrolysis assays carried out with C. reinhardtii culture supernatants, as well as the general capability of CB uptake and assimilation, indicated that this alga degrades different cellulosic substrates and internalizes resulting fragments. In summary, we conclude that C. reinhardtii converts soluble and crystalline cellulose to CB, which can be transported into the algal cell and subsequently fed into metabolism, as indicated by tritium-labelling experiments.

Sensing of exogenous cellulose induces cellulase expression

Determination of mRNA steady-state levels concerning the identified GHF9 cellulase and β-glucosidase genes implies the existence of a substrate-induced signalling mechanism for the induction of cellulolytic activity (Fig. 4). The amount of mRNAs encoding the cellulases identified in the culture supernatant (Fig. 1c) showed a 2.3- (CrCel9B) or 3.5-fold (CrCel9C) increase in the presence of FP compared with cultivation in the absence of cellulose (Fig. 4a). Interestingly, mRNA expression of the third cellulase CrCel9D could only be detected when FP was added to the medium, demonstrating a more stringent substrate-dependent regulation of this gene (Fig. 4b). Common to all three cellulase genes was that supplementation of the medium with CB had a negligible effect on the mRNA amount (Fig. 4a, MM+CB versus MM+FP). In contrast to this, mRNA expression of the two intracellularly located β-glucosidases CrBGl1/2 was increased after addition of both CB and FP, suggesting a direct correlation between substrate availability and expression induction (Fig. 4c). In summary, the data demonstrate an expression induction of the cellulolytic system including cellulases and β-glucosidases, if crystalline cellulose is present in the microenvironment of the algal cell.