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Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica.

Pinheiro GL, Correa RF, Cunha RS, Cardoso AM, Chaia C, Clementino MM, Garcia ES, de Souza W, Frasés S - Front Microbiol (2015)

Bottom Line: To overcome this hindrance, significant efforts are underway to identify novel cellulases.Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system.Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

View Article: PubMed Central - PubMed

Affiliation: Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia Rio de Janeiro, Brazil ; Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Rio de Janeiro, Brazil.

ABSTRACT
The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-β-D-cellobioside (MUC), and 4-methylumbelliferyl-β-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

No MeSH data available.


Related in: MedlinePlus

Enzymatic agar plate assay. Representative negative, positive, and double positive isolates for each substrate are shown. (A) CMC, carboxymethylcellulose; (B) Bagasse, powdered sugarcane bagasse; (C) MUG, 4-methylumbelliferyl-β-D-glucopyranoside; (D) MUC, 4-methylumbelliferyl-β-D-cellobioside; (E) pNPG, p-nitrophenyl-β-D-glucopyranoside; (F) pNPC, p-nitrophenyl-β-D-cellobioside; and (G) MUX, 4-methylumbelliferyl-β-D-xylopyranoside. For bagasse and CMC, the enzyme detection was based on the appearance of negative halo after Congo red stain. For the fluorescent MUC, MUG, and MUX, the plates were UV-irradiated. For the colorimetric substrates pNPC and pNPG, the enzymatic activity was proportional to the development of yellow color. Legends: (−), no detectable hydrolysis; (+), hydrolysis; (++), high hydrolysis. Strains were designated C to indicate isolated from crop; I, from intestine; R, from rectum. Scale bar, 1.0 cm. Note that scale bar applies to all three panels in a series. Also note that strain IDs are shown.
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Figure 3: Enzymatic agar plate assay. Representative negative, positive, and double positive isolates for each substrate are shown. (A) CMC, carboxymethylcellulose; (B) Bagasse, powdered sugarcane bagasse; (C) MUG, 4-methylumbelliferyl-β-D-glucopyranoside; (D) MUC, 4-methylumbelliferyl-β-D-cellobioside; (E) pNPG, p-nitrophenyl-β-D-glucopyranoside; (F) pNPC, p-nitrophenyl-β-D-cellobioside; and (G) MUX, 4-methylumbelliferyl-β-D-xylopyranoside. For bagasse and CMC, the enzyme detection was based on the appearance of negative halo after Congo red stain. For the fluorescent MUC, MUG, and MUX, the plates were UV-irradiated. For the colorimetric substrates pNPC and pNPG, the enzymatic activity was proportional to the development of yellow color. Legends: (−), no detectable hydrolysis; (+), hydrolysis; (++), high hydrolysis. Strains were designated C to indicate isolated from crop; I, from intestine; R, from rectum. Scale bar, 1.0 cm. Note that scale bar applies to all three panels in a series. Also note that strain IDs are shown.

Mentions: Phylogenetic relationships of the isolates together with representative 16S bacterial sequences were also analyzed (Figure 2). In order to identify the phylogenetic groups that were most efficient in degrading cellulosic compounds, their general repertoire of oligosaccharide-degrading enzymes were evaluated in parallel by enzymatic plate assays (Figure 3). The isolates were ordered by hydrolysis profile similarities and a summary is shown in Table 2. The resulting tree showed that the 40 isolates could be classified into several groups on the basis of similarities in 16S rRNA sequences. Notably, similar hydrolytic profiles could be visualized among phylogenetic-related isolates (Table 2). In the Cellulosimicrobium branch, the isolates I22A, I22B, and I37.1 were closely related to Cellulosimicrobium funkei, whereas I38C and I38D were more related to Cellulosimicrobium cellulans (Figure 2). I38E was put in a separate branch of the tree and showed only 97% of identity with C. funkei 16S rRNA sequence (Table 1). Four isolates were grouped in the Microbacterium branch. R38A and R38E were closely related to Microbacterium binotii (100 and 99% identity, respectively), whereas C8 and C16 were related, in a separate branch and to a lesser extent, to Microbacterium paraoxydans (96 and 98% identity, respectively) (Figure 2). This phylogenetic separation between R38A/R38E and C8/C16 agreed well with their cellulolytic potentials. Whereas, R38A and R38E were highly cellulolytic, as showed by the enzymatic plate assay, C8 and C16 were not capable of hydrolyzing the sugarcane bagasse or CMC, only the cello-oligosaccharides MUG, pNPG, and MUC (Table 2). The isolate R7.1 showed 97% identity with Agromyces allii strain UMS-62 16S rRNA sequence (NR_043931.1) (Table 1). Although many members of the genus Agromyces have been isolated worldwide from soil (Li et al., 2003; Jurado et al., 2005; Yoon et al., 2008; Zhang et al., 2010), their cellulolytic capacities were not reported. The isolates R7.1, R38.2, R38A, R38E, I22A, I22B, I37.1, I38C, I38D and I38E, all from Actinobacteria phylum, displayed very similar hydrolytic profiles (Table 2), being able to degrade all of the substrate tested, including the highly recalcitrant powdered sugarcane bagasse. Interestingly, all of the bagasse-degrading isolates also hydrolyzed CMC. All of the CMC- and bagasse-degrading isolates also degrade pNPG and MUG, however, five isolates (C3, C8, C10, C16, I28A, I32.1, I32.2) that secrete β-glucosidase didn't degrade CMC or bagasse.


Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica.

Pinheiro GL, Correa RF, Cunha RS, Cardoso AM, Chaia C, Clementino MM, Garcia ES, de Souza W, Frasés S - Front Microbiol (2015)

Enzymatic agar plate assay. Representative negative, positive, and double positive isolates for each substrate are shown. (A) CMC, carboxymethylcellulose; (B) Bagasse, powdered sugarcane bagasse; (C) MUG, 4-methylumbelliferyl-β-D-glucopyranoside; (D) MUC, 4-methylumbelliferyl-β-D-cellobioside; (E) pNPG, p-nitrophenyl-β-D-glucopyranoside; (F) pNPC, p-nitrophenyl-β-D-cellobioside; and (G) MUX, 4-methylumbelliferyl-β-D-xylopyranoside. For bagasse and CMC, the enzyme detection was based on the appearance of negative halo after Congo red stain. For the fluorescent MUC, MUG, and MUX, the plates were UV-irradiated. For the colorimetric substrates pNPC and pNPG, the enzymatic activity was proportional to the development of yellow color. Legends: (−), no detectable hydrolysis; (+), hydrolysis; (++), high hydrolysis. Strains were designated C to indicate isolated from crop; I, from intestine; R, from rectum. Scale bar, 1.0 cm. Note that scale bar applies to all three panels in a series. Also note that strain IDs are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542579&req=5

Figure 3: Enzymatic agar plate assay. Representative negative, positive, and double positive isolates for each substrate are shown. (A) CMC, carboxymethylcellulose; (B) Bagasse, powdered sugarcane bagasse; (C) MUG, 4-methylumbelliferyl-β-D-glucopyranoside; (D) MUC, 4-methylumbelliferyl-β-D-cellobioside; (E) pNPG, p-nitrophenyl-β-D-glucopyranoside; (F) pNPC, p-nitrophenyl-β-D-cellobioside; and (G) MUX, 4-methylumbelliferyl-β-D-xylopyranoside. For bagasse and CMC, the enzyme detection was based on the appearance of negative halo after Congo red stain. For the fluorescent MUC, MUG, and MUX, the plates were UV-irradiated. For the colorimetric substrates pNPC and pNPG, the enzymatic activity was proportional to the development of yellow color. Legends: (−), no detectable hydrolysis; (+), hydrolysis; (++), high hydrolysis. Strains were designated C to indicate isolated from crop; I, from intestine; R, from rectum. Scale bar, 1.0 cm. Note that scale bar applies to all three panels in a series. Also note that strain IDs are shown.
Mentions: Phylogenetic relationships of the isolates together with representative 16S bacterial sequences were also analyzed (Figure 2). In order to identify the phylogenetic groups that were most efficient in degrading cellulosic compounds, their general repertoire of oligosaccharide-degrading enzymes were evaluated in parallel by enzymatic plate assays (Figure 3). The isolates were ordered by hydrolysis profile similarities and a summary is shown in Table 2. The resulting tree showed that the 40 isolates could be classified into several groups on the basis of similarities in 16S rRNA sequences. Notably, similar hydrolytic profiles could be visualized among phylogenetic-related isolates (Table 2). In the Cellulosimicrobium branch, the isolates I22A, I22B, and I37.1 were closely related to Cellulosimicrobium funkei, whereas I38C and I38D were more related to Cellulosimicrobium cellulans (Figure 2). I38E was put in a separate branch of the tree and showed only 97% of identity with C. funkei 16S rRNA sequence (Table 1). Four isolates were grouped in the Microbacterium branch. R38A and R38E were closely related to Microbacterium binotii (100 and 99% identity, respectively), whereas C8 and C16 were related, in a separate branch and to a lesser extent, to Microbacterium paraoxydans (96 and 98% identity, respectively) (Figure 2). This phylogenetic separation between R38A/R38E and C8/C16 agreed well with their cellulolytic potentials. Whereas, R38A and R38E were highly cellulolytic, as showed by the enzymatic plate assay, C8 and C16 were not capable of hydrolyzing the sugarcane bagasse or CMC, only the cello-oligosaccharides MUG, pNPG, and MUC (Table 2). The isolate R7.1 showed 97% identity with Agromyces allii strain UMS-62 16S rRNA sequence (NR_043931.1) (Table 1). Although many members of the genus Agromyces have been isolated worldwide from soil (Li et al., 2003; Jurado et al., 2005; Yoon et al., 2008; Zhang et al., 2010), their cellulolytic capacities were not reported. The isolates R7.1, R38.2, R38A, R38E, I22A, I22B, I37.1, I38C, I38D and I38E, all from Actinobacteria phylum, displayed very similar hydrolytic profiles (Table 2), being able to degrade all of the substrate tested, including the highly recalcitrant powdered sugarcane bagasse. Interestingly, all of the bagasse-degrading isolates also hydrolyzed CMC. All of the CMC- and bagasse-degrading isolates also degrade pNPG and MUG, however, five isolates (C3, C8, C10, C16, I28A, I32.1, I32.2) that secrete β-glucosidase didn't degrade CMC or bagasse.

Bottom Line: To overcome this hindrance, significant efforts are underway to identify novel cellulases.Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system.Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

View Article: PubMed Central - PubMed

Affiliation: Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia Rio de Janeiro, Brazil ; Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Rio de Janeiro, Brazil.

ABSTRACT
The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), 4-methylumbelliferyl-β-D-cellobioside (MUC), and 4-methylumbelliferyl-β-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.

No MeSH data available.


Related in: MedlinePlus