Isolation and molecular identification of cellulose-degrading bacteria from rumen sheep ''Ovis aries'' and evaluation of their cellulase production

Abstract

This study focuses on the isolation and identification of cellulose-degrading bacteria from the digestive tract of sheep (Ovis aries) and to determine the cellulase capacity of different isolates for ulterior application. The bacterial strains were screened firstly using Congo red, where cellulase activity is indicated by the appearance of a hydrolysis zone on the Carboxymethylcellulose medium (CMC), the clearance zone value ranged between 8 and 15 mm for all isolates and the hydrolytic capacity was between 1.6 to 2.5 mm. The cellulolytic strain isolates were identified using MALDI-TOF Mass Spectrometry and 16S ribosomal RNA gene sequencing. MALDI-TOF MS test indicated that all strains belonged to Bacillaceae family. The strains Isolate2, Isolate3, and Isolate8 were reliable to B. tequilensis species with a score value 2.0. Whereas, the Isolate1, Isolate4, Isolate5, and Isolate6 were reliably identified to the genus level (Score value 1.7–1.99). The molecular identification results revealed that the strains indicate a high sequence similarity with 16S rRNA gene sequences accessible in GenBank database and belong to Priestia megaterium, Lysinibacillus capsici, Bacillus tequilensis, and Bacillus paralicheniformis. The highest Carboxymethylcellulase activity (CMCase) was obtained by B. tequilensis (0.827 ± 0.035 U/ml) at a pH of 7.0 and temperature of 30 °C at 100 g. The carbon source utilized was CMC (1 %), while peptone (1 %) and ammonium sulfate (0.24 %) served as the nitrogen sources. Further research in optimizing and purifying of cellulase, could be useful for the future hydrolyzation of some green biomass for various biotechnological applications such as biofuel production.

Keywords

• Bacillaceae
• 16S-rRNA
• MALDI-TOF MS
• Cellulase
• Cellulolytic bacteria
• CMCase