Computational protocol: Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials

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Protocol publication

[…] Twenty-four bacterial strains, belonging to the microbial collection of the Department of Agricultural Sciences, division of Microbiology of the University of Naples Federico II, were used in this study. These strains, previously isolated from lignocellulosic biomass of A. donax, E. camaldulensis, and P. nigra during biodegradation under natural conditions (Ventorino et al., ), were identified as Actinobacteria on the basis of their colony morphology, microscopic features (phase-contrast microscopy, shape, dimension, and presence of spores) and biochemical characteristics (Gram-stains, catalase activity).Molecular identification was performed by 16S rRNA gene sequencing. Fast DNA SPIN kit for soil (MP Biomedicals, Illkirch Cedex, France) was used to extract and purify the total genomic DNA according to the supplier's recommendations. Approximately 50 ng of DNA was used as template for PCR assays. Synthetic oligonucleotide primers fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rD1 (5′-AAGGAGGTGATCCAGCC-3′) were used to amplify the 16S rRNA gene as reported by Palomba et al. (). The PCR conditions were as described by Ventorino et al. (). Amplicons were purified using the QIA quick gel extraction kit (Qiagen S.p.A., Milan, Italy) after visualization by agarose (1.5% wt/vol) gel electrophoresis at 100 V for about 1 h. The DNA sequences were determined and analyzed as previously reported (Pepe et al., ), and were compared to the 16S ribosomal RNA sequences database of GenBank nucleotide data library using the BLAST software at the National Centre of Biotechnology Information website (http://www.ncbi.nlm.nih.gov/Blast.cgi). Multiple nucleotide alignments of the nearly full-length 16S rRNA sequences of bacterial isolates and type strains within each of the defined species were carried out using the ClustalW program from MEGA version 4.0 (Tamura et al., ). The nucleotide sequences of the type strains were recovered from the Ribosomal Database Project (RDP—https://rdp.cme.msu.edu). The phylogenetic tree was inferred by the Neighbor-Joining method with the Maximum Composite Likelihood model in MEGA4 program with bootstrap values based on 1000 replications. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Eucalyptus camaldulensis, Streptomyces flavogriseus, Streptomyces flavovirens
Chemicals Glucose, Xylose