Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces (S.) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies, and Streptomyces flavofuscus), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus, and Streptomyces ambofaciens. Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers (S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and xylanase preparations from Genencore (Accellerase BG and Accellerase XY). Cellulose and xylan conversion, when conducted using commercial (hemi)cellulases, gave glucose and xylose yields of 30.17 and 68.9%, respectively. The replacement of the cellulolytic preparation from Genencor (Accellerase 1500), with the endo-cellulase from S. argenteolus AE58P resulted in almost 76% of the glucose yield obtained in the presence of the commercial counterpart. Due to the promising results obtained by using the enzymatic crude extracts from S. argenteolus AE58P in the pretreated A. donax saccharification experiments, the proteins putatively responsible for endo-cellulase activity in this strain were identified by proteomics. Several proteins were confidently identified in different Streptomyces spp., eight of which belong to the class of Carbohydrate active enzymes. Overall results highlighted the biotechnological potential of S. argenteolus AE58P being an interesting candidate biocatalyst-producing bacterium for lignocellulose conversion and production of biochemicals and bioenergy.

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BackgroundBiofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels.ResultsThe selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24–72 h range.ConclusionsThe very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0569-z) contains supplementary material, which is available to authorized users.