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Additional file 2: Figure S2. Lack of correlation between ethyl acetate formation and predicted strength of the RBS controlling the production of K. marxianus trEat1. Ethyl acetate titres were obtained from E. coli BW25113 ΔackAΔldhA (DE3) producing K. marxianus trEat1 variants at 0.01 mM IPTG concentration (Fig. 2b). The translation initiation rates of the RBS were predicted with the RBS calculator [22].

Additional file 1. Additional figures and tables.

Additional file 1: Table S1. Overview of pH-controlled batch fermentations in 1.5L Applikon bioreactors with continuous gas stripping. Measured and calculated concentrations of main fermentation products, carbon balance and C-mol yields at end of fermentations are represented as average (AV) with standard deviations (SD) for each duplicate. E. coli BW25113 ΔackAΔldhA (DE3) producing Eat1 variants from pET26b plasmids were grown under anoxic conditions in minimal medium containing 55 mM glucose. Expression of Eat1 was induced by IPTG.

Additional file 1. Supplementary file.

Figure S1. Phylogenetic tree of GH5 family hydrolases. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [42]. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Jones Thornton Taylor (JTT) model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 27 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 198 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [43]. Open circle: eukaryotic origin; closed triangle: thermophilic organism; closed diamond: sequences obtained in this study. (PDF 12 kb)

Table S2. Overview of measured absorbance of different chromogenic substrates after incubation with the metagenome derived putative cellulases GE39 and GE40 expressed from E. coli. Overview of measured absorbance of different chromogenic substrates after incubation with the metagenome derived putative cellulases GE39 and GE40 expressed from E. coli. The final reaction mixture in each well of the substrate plate consisted of 145 μL sodium phosphate buffer (pH 6.0) and 5 μL of CFE. Plates were then sealed using an aluminum adhesive foil and incubated at 60 °C in a rotary shaker at 180 RPM. After 24 h the reaction mixture was collected in a product plate by centrifugation (2700×g, 10 min) and absorbance was measured at 595 nm (blue) and 517 nm (red) using a plate reader (Biotek Instruments Inc., Winooski, VT, USA). Negative control consisted of sodium phosphate buffer and CFE from an E. coli culture containing empty pCDF1b plasmid. The thermostable endoglucanase, CelTM, (Megazyme, Wicklow, Ireland) from Thermotoga maritima was used as positive control (+C) at a concentration of 1 μg/mL. Values for the negative control have been subtracted. CFE of the GE40 expressing E. coli culture showed high activity towards cellulose and barley derived β-glucan. In contrast, CFE of the GE39 producing E. coli culture showed no activity to any of the chromogenic substrates. (PDF 147 kb)

Figure S2. Congo red assays of cellulase expressing G. thermodenitrificans cultures. Congo red assays of G. thermodenitrificans cultures grown on LB2 medium with 1% carboxymethylcellulose. Each culture produces a different cellulase. Ă : empty plasmid (pNW33n) control; CelA: GH5 endoglucanase CelA (Geobacillus 70PC53); CelK: GH9 exoglucanase (C. thermocellum); GE40wt: GH5 endoglucanase (Geobacillus metagenome derived) containing its native signal peptide; GE40: GH5 endoglucanase (Geobacillus metagenome derived). (PDF 48 kb)

10% SDS-PAGE of purified endo-xylanase from G. thermodenitrificans T12 followed by PageBlue staining. Lane 1: Protein marker; Lane 2: Pellet fraction; Lane 3: cell-free extract; Lane 4: non-binding protein fraction from FPLC; Lane 5: Purified recombinant GtXynA1; Lane 6: Purified and desalted recombinant GtXynA1; Lane 7: Protein marker. (DOCX 614 kb)

FPLC purification of GtXynA1. Protein fraction not bound to the nickel column eluted with the first 65 mL eluent. Bound GtXynA1 protein was removed from the nickel column using a imidazole gradient (yellow line) which increased from 0 mM to 500 mM over a time span of 20 min (A). Fractions 22–25 were pooled and used for desalting the purified GtXynA1 (B). Fractions 8–10 of the desalting column were pooled and then used for further experiments. (DOCX 311 kb)

Additional file 2: Table S1. Overview of the number of isolates recovered from the first isolation. Overview of the number of isolates recovered from the first isolation on CTVMy-CMC and CTVM-CMC media after 20 h, 48 h and 88 h. The pH was set at 6.5 and plates were incubated at 65 °C. Dilution series were plated from compost that had been shaken at 150 rpm for 3 h in the same medium and temperature.

Additional file 4: Table S3. HPLC data from isolates ranked on total organic acid production and total lactic acid production on xylose (C5).

Additional file 3: Table S2. HPLC data from isolates ranked on total organic acid production and total lactic acid production on cellobiose (C6).

This file shows the original gel for Figure 2 C. It is the same gel as in Figure A-B/additional file 1, but with a longer exposure time.

Fermentation analysis of B. smithii ET 138 wild-type and mutant strains in 1Â L pH-controlled bioreactors after Âą24Â h. This table shows fermentation data in pH-controlled lab-scale reactors to supplement the data in tubes described in the main text.

This file shows the original gel for Figure 2A and B. It is the same gel as in Figure C/additional file 2, but with a shorter exposure time.

B. smithii ΔldhL-ΔsigF after counter-selection on plates containing 100 μg/mL X-gal. This figure shows the difference in colony size on 100 µg/mL X-gal after lacZ counter-selection.