Cellulases

The medium used for the second screening step using the Congo red test was similar to that described above (Section 2.2), except that the carbon source was low viscosity carboxymethylcellulose (CMC) (Sigma, USA). Only those strains that showed substantial growth in the initial screening with Avicel were selected for the Congo red test. Inoculation was carried out by using a platinum needle to transfer the spores from the PDA plate to the center of the plates containing the CMC medium [18 ]. The inoculated plates were incubated for 96 h at 30°C and the growth of the microorganism was measured by the diameter of the colony. A 10 mL aliquot of Congo red dye (2.5 g·L⁻¹) was then added to each plate. After 15 min, the solution was discarded and the cultures were washed with 10 mL of 1 mol·L⁻¹ NaCl. Cellulase production was indicated by the appearance of a pale halo with orange edges, indicative of areas of hydrolysis. This halo was measured for subsequent calculation of the enzymatic index (EI) using the expression:
$\begin{array}{c}\text{EI}=\frac{\text{diameter}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{of}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{hydrolysis}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{zone}}{\text{diameter}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{of}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{colony}}.\end{array}$
The strains that showed an EI higher than 1.50 were considered to be potential producers of cellulases. Three independent experiments were performed for this screening step, with two replicates per strain. For each strain the average EI of the three experiments was calculated, together with the standard deviation.

Genomic DNA extracted from the day 31 sample was used for sequencing library construction following the DOE Joint Genome Institute standard operating procedure for shotgun sequencing using the Roche 454 GS FLX Titanium technology. Obtained sequencing reads were quality trimmed and assembled using the Newbler assembler software (version 2) by 454 Life Sciences. For assembly, minimum acceptable overlap match (mi) was set to 0.95. Quality filtered sequence reads and assembled contigs ≥100 bp totaling 110 Mbp were used for further analysis. For global functional analysis, the metagenomic data set was loaded into MG-RAST [37] (link) and compared to other annotated metagenomes that are publicly available in the metagenome analysis platform. Correspondence analysis was performed using the R software package ade4 [38] .
Glycoside hydrolases of selected functional classes (e.g. cellulases, endohemicellulases, debranching enzymes) were identified using pfam HMMs (Pfam version 23.0 and HMMER v2.3). For the 3 GH families 44, 51 and 74 that are not represented in pfam, HMMs were generated (two for each, since they are 2-domain proteins) and treated similar to the pfam HMMs. For GH families covered by multiple pfams (e.g. GH2 or GH42) only the best scoring hit was taken into account in case there were multiple hits to the same contig. Contig read depth was factored as following: based on the Newbler output, the number of reads in each was determined and multiplied by the median read length of 400 bp and divided by the contig length. This weighting corrects approximately for differences in species abundance distribution (i.e. dominant populations producing higher depth contigs will be weighted in the analysis).
To extract potential full-length glycoside hydrolases from the metagenome data, we ran BLASTX on all contigs ≥1 kb against the CAZy [3] (link) and FOLy [39] (link) databases (E<1e⁻¹⁰), and filtered out hits matching the target enzyme over at least 90% of its length, and for which the target enzyme has a known enzymatic function (EC number listed in CAZy or FOLy). Frameshifts (most likely introduced by homopolymers during sequencing) were delineated by BLASTX of the targeted contigs against the non-redundant NCBI nucleotide database and corrected by deleting or duplicating single bases so as to maximize the BLAST scores. After manual frameshift correction, genes were called using fgenesb (http://www.softberry.com). For phylogenetic analysis, peptide sequences of the two full-length GH9 enzymes were aligned to reference sequences using ClustalX [40] (link) and imported into the ARB software package [41] (link) for phylogenetic reconstructions using the PROML function of the integrated Phylip package.

The CS was collected from local farm (Yuanyang county, Henan province, China, 113.9^oE, 35.1^oN), and then totally dried, ground and screened with a 60 mesh sieve. The pulping was carried out by cooking with 2% (w/v) NaOH solution at 80°C and atmospheric pressure for 2 h. The ratio of CS to NaOH was 1:20 (w/w). Wash the pulp with pure water until pH maintain stable, then totally dry the pulp in oven at 105°C. The hydrolysis was carried out with cellulases (Qingdao Vland Biotech Inc., Qingdao, China) in deionized water (adjust to pH5.0 with acetic acid). It should be noted that the hydrolysis buffer should not contain any sodium ion, which will cause the calcium alginate fiber instable in the next step. A final concentration of 10% (w/v) substrate and (10 FPU cellulase)/(g substrate) was added. Incubate the mixture at 50°C and 200 rpm for 3 days for sufficient hydrolysis.

The MWLs from macaúba, carnauba, and coconut fruit endocarps were isolated using the experimental conditions previously described (13 (link), 68 (link)). Briefly, ~40 g of extractive-free samples was ball-milled in a PM100 mill (Restch, Haan, Germany) for 6 hours, at 400 rpm, in a 500-ml agate jar and using agate ball bearings (20 × 20 mm). The finely powdered samples were extracted with 1 liter of dioxane:water 96:4 (v/v) with stirring in the dark for 24 hours. The solution was centrifuged, and the supernatant was collected by decantation. The extraction process was repeated two more times, using fresh dioxane:water each time, and the collected supernatants were combined and evaporated to dryness on a rotary evaporator at 40°C. The residue obtained (crude lignin) was then purified as described elsewhere (68 (link)). The final yields were about 15% of the Klason lignin. The lignin preparation obtained in this way, known as “milled wood lignin”, preserves intact the main structural characteristics of lignin in its native form (69 ).
The ELs were prepared from extractive-free cell walls as detailed in a previous publication (70 (link)). Briefly, the extractive-free ball-milled cell walls were treated with cellulases (Cellulysin, EC 3.2.1.4; activity, >10,000 units/g; Calbiochem) from Trichoderma viride. The cell walls (450 mg) were suspended in NaOAc buffer (pH 5), and 22.5 mg of Cellulysin was added. The reaction mixture was shaken on a rotary incubator shaker at 35°C for 48 hours. The residue was collected by centrifugation (8000 rpm, 30 min), and the enzyme digestion process was repeated three times. The collected residue was sonicated and washed with deionized water (20 ml) three times and lyophilized. The collected EL contents were 56.3 and 53.8% for macaúba and carnauba, respectively (Table 2B).

In sequential addition of cellulases, beginning with the addition of Endo-1,4- $\mathrm{\beta }$ -glucanase (200 U) to the screw-capped reagent bottle enclosing the pre-treated substrate i.e., halophyte Atriplex crassifolia (0.25 g), the reaction was carried out in a shaking water bath set at 75°C and 50 rpm. After 2 h of incubation time, Exo-1,4- $\mathrm{\beta }$ -glucanase (400 U) was added to the same reagent bottle for another 2 h. Next, the cellulase i.e., $\mathrm{\beta }$ -1,4-glucosidase was added to the same reagent bottle and incubated for a period of 2 h. In this way, in sequential addition of cellulases, each cellulase was added one after the other and each was incubated for a period of 2 h.