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Isolation of cellulolytic bacteria from the intestine of Diatraea saccharalis larvae and evaluation of their capacity to degrade sugarcane biomass.

Dantur KI, Enrique R, Welin B, Castagnaro AP - AMB Express (2015)

Bottom Line: Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella.Good cellulolytic activity correlated with high extracellular protein concentrations.In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates.

View Article: PubMed Central - PubMed

Affiliation: Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA), 3150 William Cross Av., Las Talitas, PC T4101XAC Tucumán Argentina.

ABSTRACT
As a strategy to find efficient lignocellulose degrading enzymes/microorganisms for sugarcane biomass pretreatment purposes, 118 culturable bacterial strains were isolated from intestines of sugarcane-fed larvae of the moth Diatraea saccharalis. All strains were tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays or by growing bacteria on sugarcane biomass as sole carbon sources. Out of the 118 strains isolated thirty eight were found to possess cellulose degrading activity and phylogenetic studies of the 16S rDNA sequence revealed that all cellulolytic strains belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. Within the three phyla, species belonging to five different genera were identified (Klebsiella, Stenotrophomonas, Microbacterium, Bacillus and Enterococcus). Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella. Good cellulolytic activity correlated with high extracellular protein concentrations. In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates. The results of this study indicate the possibility to find efficient cellulose degrading enzymes and microorganisms from intestines of insect larvae feeding on sugarcane and their possible application in industrial processing of sugarcane biomass such as second generation biofuel production.

No MeSH data available.


Related in: MedlinePlus

Measurement of extracellular endo-glucanase activity. A) Enzyme activity tested through hydrolysis of CMC by supernatants of bacteria grown on CMC and bagasse. Cell and fiber-free supernatants were incubated for 10 days at 30°C before staining with Congo red. Halos around wells indicate extracellular endo-glucanase activity. B) Total extracellular protein concentration as measured by the method of Bradford of bacterial cultures incubated in medium containing sugarcane residues or CMC. C) CMCase activity in supernatants of bacterial strain selected for high cellulolytic activity grown on CMC and bagasse expressed in units.ml-1. Bacterial abbreviations: E = Enterococcus casseliflavus Kd7 TUC-EEAOC, K = Klebsiella oxytoca Kd70 TUC-EEAOC and B = Bacillus pumilus Kd109 TUC-EEAOC, S = Stenotrophomonas maltophilia Kd3 TUC-EEAOC.
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Fig4: Measurement of extracellular endo-glucanase activity. A) Enzyme activity tested through hydrolysis of CMC by supernatants of bacteria grown on CMC and bagasse. Cell and fiber-free supernatants were incubated for 10 days at 30°C before staining with Congo red. Halos around wells indicate extracellular endo-glucanase activity. B) Total extracellular protein concentration as measured by the method of Bradford of bacterial cultures incubated in medium containing sugarcane residues or CMC. C) CMCase activity in supernatants of bacterial strain selected for high cellulolytic activity grown on CMC and bagasse expressed in units.ml-1. Bacterial abbreviations: E = Enterococcus casseliflavus Kd7 TUC-EEAOC, K = Klebsiella oxytoca Kd70 TUC-EEAOC and B = Bacillus pumilus Kd109 TUC-EEAOC, S = Stenotrophomonas maltophilia Kd3 TUC-EEAOC.

Mentions: Endo-glucanases cleave amorphous regions of the cellulose microfibril producing termini of cellulose chains whereas exo-glucanases act on these termini to loosen the crystalline structure of the microfibril. Endo- and exo-glucanase activities are measured using soluble substrates (CMC) and crystalline forms of cellulose (alpha-cellulose or Avicel), respectively. Exo- and endo-glucanase activity was analyzed for the thirty-eight (38) bacterial isolates selected using agar plates containing either bagasse, HT or CMC as carbon sources. After 10 days of growth at 30°C, bacterial plates were stained with Congo red and scored for presence of a clearing zone (halo) around each colony, indicating cellulose degradation. Among the isolates tested, only strains belonging to species of the genera Klebsiella and Bacillus demonstrated clear total cellulase and endo-glucanase activities (Figure 3A, 3B, 3C and Table 1). Bacteria belonging to species of the genera Enterococcus and Stenotrophomonas showed marked endo-glucanase activity on CMC plates (Figures 3C and 4A) but despite that they were able to grow on lignocellulose substrates did not show any noticeable clearing zones of hydrolysis on sugarcane biomass substrates (Figures 3A, 3B and 4A). Bacterial strains of species belonging to the genus Microbacterium displayed a very low cellulolytic activity when grown on CMC plates and were unable to grow on any of the two lignocellulose substrates from sugarcane tested (Figure 3A and Figure 3B).Figure 3


Isolation of cellulolytic bacteria from the intestine of Diatraea saccharalis larvae and evaluation of their capacity to degrade sugarcane biomass.

Dantur KI, Enrique R, Welin B, Castagnaro AP - AMB Express (2015)

Measurement of extracellular endo-glucanase activity. A) Enzyme activity tested through hydrolysis of CMC by supernatants of bacteria grown on CMC and bagasse. Cell and fiber-free supernatants were incubated for 10 days at 30°C before staining with Congo red. Halos around wells indicate extracellular endo-glucanase activity. B) Total extracellular protein concentration as measured by the method of Bradford of bacterial cultures incubated in medium containing sugarcane residues or CMC. C) CMCase activity in supernatants of bacterial strain selected for high cellulolytic activity grown on CMC and bagasse expressed in units.ml-1. Bacterial abbreviations: E = Enterococcus casseliflavus Kd7 TUC-EEAOC, K = Klebsiella oxytoca Kd70 TUC-EEAOC and B = Bacillus pumilus Kd109 TUC-EEAOC, S = Stenotrophomonas maltophilia Kd3 TUC-EEAOC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385043&req=5

Fig4: Measurement of extracellular endo-glucanase activity. A) Enzyme activity tested through hydrolysis of CMC by supernatants of bacteria grown on CMC and bagasse. Cell and fiber-free supernatants were incubated for 10 days at 30°C before staining with Congo red. Halos around wells indicate extracellular endo-glucanase activity. B) Total extracellular protein concentration as measured by the method of Bradford of bacterial cultures incubated in medium containing sugarcane residues or CMC. C) CMCase activity in supernatants of bacterial strain selected for high cellulolytic activity grown on CMC and bagasse expressed in units.ml-1. Bacterial abbreviations: E = Enterococcus casseliflavus Kd7 TUC-EEAOC, K = Klebsiella oxytoca Kd70 TUC-EEAOC and B = Bacillus pumilus Kd109 TUC-EEAOC, S = Stenotrophomonas maltophilia Kd3 TUC-EEAOC.
Mentions: Endo-glucanases cleave amorphous regions of the cellulose microfibril producing termini of cellulose chains whereas exo-glucanases act on these termini to loosen the crystalline structure of the microfibril. Endo- and exo-glucanase activities are measured using soluble substrates (CMC) and crystalline forms of cellulose (alpha-cellulose or Avicel), respectively. Exo- and endo-glucanase activity was analyzed for the thirty-eight (38) bacterial isolates selected using agar plates containing either bagasse, HT or CMC as carbon sources. After 10 days of growth at 30°C, bacterial plates were stained with Congo red and scored for presence of a clearing zone (halo) around each colony, indicating cellulose degradation. Among the isolates tested, only strains belonging to species of the genera Klebsiella and Bacillus demonstrated clear total cellulase and endo-glucanase activities (Figure 3A, 3B, 3C and Table 1). Bacteria belonging to species of the genera Enterococcus and Stenotrophomonas showed marked endo-glucanase activity on CMC plates (Figures 3C and 4A) but despite that they were able to grow on lignocellulose substrates did not show any noticeable clearing zones of hydrolysis on sugarcane biomass substrates (Figures 3A, 3B and 4A). Bacterial strains of species belonging to the genus Microbacterium displayed a very low cellulolytic activity when grown on CMC plates and were unable to grow on any of the two lignocellulose substrates from sugarcane tested (Figure 3A and Figure 3B).Figure 3

Bottom Line: Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella.Good cellulolytic activity correlated with high extracellular protein concentrations.In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates.

View Article: PubMed Central - PubMed

Affiliation: Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA), 3150 William Cross Av., Las Talitas, PC T4101XAC Tucumán Argentina.

ABSTRACT
As a strategy to find efficient lignocellulose degrading enzymes/microorganisms for sugarcane biomass pretreatment purposes, 118 culturable bacterial strains were isolated from intestines of sugarcane-fed larvae of the moth Diatraea saccharalis. All strains were tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays or by growing bacteria on sugarcane biomass as sole carbon sources. Out of the 118 strains isolated thirty eight were found to possess cellulose degrading activity and phylogenetic studies of the 16S rDNA sequence revealed that all cellulolytic strains belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. Within the three phyla, species belonging to five different genera were identified (Klebsiella, Stenotrophomonas, Microbacterium, Bacillus and Enterococcus). Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella. Good cellulolytic activity correlated with high extracellular protein concentrations. In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates. The results of this study indicate the possibility to find efficient cellulose degrading enzymes and microorganisms from intestines of insect larvae feeding on sugarcane and their possible application in industrial processing of sugarcane biomass such as second generation biofuel production.

No MeSH data available.


Related in: MedlinePlus