The bioconversion of cellulose and hemicellulose to soluble sugars is important for global stabilization and a sustainable human society. Here, hundreds of cellulolytic bacteria were screened and isolated from soil, compost, and animal waste slurry in Jeju Island, South Korea. Among the isolates, three strains, SL9-9, C5-16, and S52-2, showing higher potential for practical uses were purified on carboxymethyl cellulose (CMC) agar plates and identified as
The bioconversion of cellulose to soluble sugars and glucose is catalyzed by a group of enzymes called cellulases that are produced by microorganisms [
These cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant and renewable biopolymer on Earth. The demand for microbial cellulases and related enzymes is growing more rapidly than ever before [
Fungal cellulases are produced in large amounts, which include all the components of a multienzyme system with different specificities and modes of action, that is, endoglucanases (or CMCase), exoglucanases (cellobiohydrolase), and
Synergic multienzyme systems are also expected in bacterial cellulase complexes. Cellulolytic bacteria include aerobes such as
We isolated cellulase-producing
Three strains,
A total of 176 samples were collected from soil, compost, and animal waste slurry on Jeju Island, South Korea, and were screened for cellulolytic bacteria. The samples were stored at 4°C in the dark until use. After appropriate dilutions with sterile water, 1 mL each of the sample dilutions was spread onto carboxymethyl cellulose (CMC) agar plates that consisted of CMC, 10.0; yeast extract, 1.0; (NH4)2SO4, 2.5; K2HPO4
The isolates were morphologically and physiologically characterized and identified up to genus level according to Bergey's Manual of Determinative Bacteriology (8th edition). An API 50 CHB kit (BioMérieux, Lyon, France) was also used for the identification of Gram-positive bacteria. API strips were inoculated with 24 h-grown cultures and then incubated at 28°C. The results were read according to the manufacturer's instructions and compared with other known
Genes of 16S rRNA were sequenced and compared for identification of the bacterial isolates. The bacterial cells grown on CMC agar were harvested and used for chromosomal DNA isolation according to the protocols [
Starter cultures were prepared by transferring cells with an inoculation loop from the CMC agar plates to 100 mL of CMC liquid medium, the initial pH of which was adjusted to 7.0 if not specified, in 500 mL Erlenmeyer flasks. Two days after shaking incubation at 28°C, aliquots of 2 mL starter cultures were seeded into 200 mL of CMC liquid medium in 500 mL flasks. The flasks were further incubated on a shaker at 150 rpm for 7 days at 28°C. Cell growth was monitored by measuring optical density at 600 nm. Culture samples were taken every 24 h during incubation, and their cell-free supernatants (CFSs) were obtained by centrifugation (10,000 ×g, 5 min) and analyzed for cellulolytic activities. Meanwhile, the precipitated cells were suspended, washed in 5 mL of 0.05 M phosphate buffer (pH 6.5), and disrupted by sonication (150 mA, 20 min). The resulting supernatant was removed after centrifuging (12,000 ×g, 30 min) the sonicated cell suspension at 4°C. The remaining cell debris (CD) was resuspended in 1 mL of 0.05 M phosphate buffer (pH 6.5) and assayed for cellulolytic enzyme activities.
CMCase activity was measured by incubating 0.2 mL of enzyme solution with 0.5 mL of 1% (w/v) carboxymethyl cellulose, prepared in 0.1 M sodium acetate buffer (pH 5.0), and 0.3 mL of 0.1 M sodium acetate buffer (pH 5.0) for 10 min at 50°C. The reducing sugars liberated were estimated by the 3,5-dinitrosalicylic acid (DNS) method [
Avicelase activity was measured by incubating 0.5 mL of enzyme solution with 1 g of Avicel, a microcrystalline cellulose, as substrate and 1.5 mL of 0.1 M sodium acetate buffer (pH 5.0) for 1 h at 50°C. After incubation, the reaction mixture was centrifuged at 10,000 ×g for 5 min, and then 1 mL of the supernatant was taken to determine reducing sugars by the DNS method. One unit of Avicelase activity was defined as the amount of enzyme that released 1
Filter paper-hydrolytic (FPase) activity was measured by a procedure [
Xylanase activity was measured by using Beechwood xylan as a substrate [
Cellulolytic bacteria were sought among 176 different samples collected from various environments such as soil, compost, and animal waste slurry on Jeju Island. Appropriate dilutions of each sample were placed on CMC agar plates. Positive clones showing good colonial development and a visible clearing zone were transferred to fresh CMC plates. A total of 309 positive clones were thus selected in the first round of screening. Their cellulolytic activities were confirmed by the trypan blue-staining method on CMC agar medium (Figure
Bacterial cell growth and CMCase activity on CMC agar plates containing trypan blue. SL9-9, isolate from animal waste slurry; C5-16, isolate from compost; S52-2, isolate from soil; 10111,
Comparison of carboxymethyl cellulase activity between
Morphological and cultural studies revealed that all the clones were Gram-positive and rod-shaped bacteria (Table
Morphological and physiological properties of the isolated cellulolytic bacteria.
Characteristics | Bacterial isolates | ||
SL9-9 | C5-16 | S52-2 | |
Gram staining | Positive (+) | Positive (+) | Positive (+) |
Motility | + | + | + |
Catalase | + | + | + |
Cell shape | Rod | Rod | Rod |
Size (L, | 2.5~3.0 | 2.5~3.0 | 2.5~3.0 |
Colony appearance | |||
Shape | Round | Round | Round |
Margin | Entire | Entire | Undulate |
Elevation | Umbonate | Umbonate | Umbonate |
Growth temp range (°C)a | 15~50 | 15~55 | 15~50 |
Growth pH rangeb | 5~10 | 4~9 | 5~9 |
aIncubated for 72 h; bIncubated for 48 h.
Biochemical properties of the isolated cellulolytic bacteria.
Testa | Strains | ||||
SL9-9 C5-16 S52-2 | KACC 10111 | KCTC 3560 | |||
Control | − | − | − | − | − |
Glycerol | |||||
Erythritol | − | − | − | − | − |
L-arabinose | |||||
D-ribose | |||||
D-xylose | v | v | v | ||
L-xylose | − | − | − | − | − |
D-adonitol | − | − | − | − | − |
D-glucose | |||||
D-fructose | |||||
D-mannose | |||||
L-sorbose | − | − | − | − | − |
L-rhamnose | − | − | − | − | − |
Dulcitol | − | − | − | − | − |
Inositol | |||||
D-mannitol | |||||
D-sorbitol | |||||
Methyl- | − | ||||
N-acetyl-glucosamine | − | − | − | − | − |
Amygdalin | − | v | |||
Arbutin | |||||
Esculin ferric citrate | |||||
Methyl- | − | − | − | − | − |
D-lactose (bovine origin) | − | − | − | ||
D-arabinose | − | − | − | − | − |
Salicin | − | ||||
D-cellobiose | |||||
D-maltose | − | ||||
D-melibiose | v | ||||
D-saccharose | |||||
D-trehalose | |||||
Inulin | − | − | v | ||
D-melezitose | − | − | − | − | − |
Glycogen | − | ||||
Xylitol | − | − | − | − | − |
Gentiobiose | − | v | − | − | v |
D-turanose | − | − | v | ||
D-lyxose | − | − | − | − | − |
D-tagatose | − | − | − | − | − |
D-fucose | − | − | − | − | − |
L-fucose | − | − | − | − | − |
D-arabitol | − | − | − | − | − |
Potassium gluconate | − | − | − | ||
Potassium2-ketogluconate | − | − | − | − | − |
D-galactose | − | − | − | − | − |
D-raffinose | v | ||||
Potassium5-ketogluconate | − | − | − | − | − |
Methyl- | − | − | − | − | − |
AmiDon (Starch) | − | ||||
L-arabitol | − | − | − | − | − |
aAPI 50CHB Kit (BioMérieux, France) was used to determine positive (+) or negative (−); v: variable.
These three bacterial isolates were finally identified by 16S rRNA gene sequence analysis. Their sequences were entered into the nucleotide-nucleotide BLAST (NCBI) system, and percentage identities were established. The highest identity for the isolate SL9-9 (accession no. HQ236379) was 99% with the
The three isolates were examined for CMCase, Avicelase,
Figure
Carboxymethyl cellulase activity in cell-free culture supernatant (a) and cell debris (b) of isolated
Figure
Avicelase activity in cell-free culture supernatant (a) and cell debris (b) of isolated
On a whole, SL9-9 presented higher activity than the other isolates from the beginning of cultivation. In the cell debris fraction, there was no definite Avicelase activity (Figure
Figure
The
Xylanase activity in cell-free culture supernatant of isolated
Three cellulolytic bacterial strains, SL9-9, C5-16, and S52-2, were isolated and identified as