Isolation and Characterization of Cellulase Producing Bacterial Strains from an Amazonian Geothermal Spring in Peru
Yolanda Cortez
Laboratorio de Micología y Biotecnología, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Peru.
Sujay Paul
Laboratorio de Micología y Biotecnología, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Peru.
Gretty K. Villena
Laboratorio de Micología y Biotecnología, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Peru.
Marcel Gutiérrez- Correa *
Laboratorio de Micología y Biotecnología, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Peru.
*Author to whom correspondence should be addressed.
Abstract
Aim: Aguas Calientes (AC) is an isolated geothermal spring located deep into the Peruvian Amazon rainforest. The principal aim of this study was to isolate and characterize thermophilic lignocellulolytic bacterial strains from AC.
Study Design: Bacterial strains were cultured in laboratory and screened for cellulase activity. Superior lignocellulolytic strains were characterized by 16S rRNA sequences.
Place and Duration of Study: Samples were collected from Aguas Calientes (7°21'12'' S, 75°00'54'' W). The duration of the study was 2013-2016.
Methodology: Primary isolation of lignocelluloytic bacterial strains was carried out by direct plating, filtration and water enrichment analysis techniques. Secondary screening was performed by semi quantitative plate clearing assay. In both primary and secondary screening assay the growth media contained carboxymethylcellulose (CMC). CMC hydrolysis by bacterial colonies was evaluated by Congo red solution. Selected strains with higher cellulase activity were characterized by 16s rRNA gene sequencing.
Results: A total of 22 best performing strains were finally selected from 110 cellulolytic bacterial strains isolated in this study. Thirteen strains were selected from water sample (CW) while 9 selected from soil sample (CS). Water isolate LMB-AC10 showed highest cellulase activity (9.5 U ml-1) at pH 7.4 among all selected strains while LMB-AC3 was found to be the most consistent one (activity was between 6.5 U ml-1 to 7.5 U ml-1) throughout different alkaline pH levels. In CS, the highest cellulase activity (5.2 U ml-1) was showed by strain LMB-SC4 at pH 7.4. Sequence homology analysis of selected bacterial 16S RNA revealed that most of our strains are similar with different reported strains of Geobacillus, more precisely with Geobacillus thermoleovorans and Geobacillus kaustophilus.
Conclusion: For the first time several thermophilic lignocellulolytic bacterial strains were isolated from a remote Amazonian geothermal spring and some of them were found very promising.
Keywords: Peruvian Amazon rainforest, geothermal spring, cellulolytic bacteria, thermophiles,, endoglucanase.