Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 4 2026 08 04 561 573 Prakriti Jhilta Department of Microbiology, Central University of Haryana, Mahendergarh, Haryana, India Vikram Poria Department of Microbiology, Central University of Haryana, Mahendergarh, Haryana, India Arjun Singh ICAR-Central Soil Salinity Research Institute, RRS Lucknow, Uttar Pradesh, India Vishal Somvanshi Division of Nematology, ICAR-Indian Agricultural Research Institute, New Delhi, India Ram Karan Department of Microbiology, University of Delhi, South Campus, New Delhi, India Surender Singh Department of Microbiology, Central University of Haryana, Mahendergarh, Haryana, India 2025 08 14 2026 05 24 Background and Objectives: Second-generation bioethanol, a sustainable and environmentally friendly alternative to fossil fuels, can significantly contribute to the economy. However, saccharification accounts for approximately 20-25% of total bioethanol production costs, which can be reduced by using the improved enzyme cocktails. Therefore, the present study aimed to isolate and characterize potent cellulolytic Streptomyces strains capable of enhancing the hydrolytic conversion of lignocellulosic biomass. Materials and Methods: Degraded wood and soil samples were collected and enriched with paddy straw. The isolates were obtained using standard methods and characterized by 16S rRNA sequencing. The best isolate was subjected to whole-genome sequencing to identify hydrolytic genes involved in agro-waste degradation. Saccharification assays were performed using crude culture filtrate in combination with the commercial cellulase cocktail Celluclast® to evaluate sugar release from alkali-pretreated paddy straw. Results: Among the 38 isolates obtained, five were identified as potential cellulolytic Streptomyces strains based on 16S rRNA gene sequencing: Streptomyces tunisiensis SSPJ1, Streptomyces griseoincarnatus SSPJ4, Streptomyces werraensis SSPJ14, Streptomyces ardesiacus SSPJ32, and Streptomyces tendae SSPJ48. S. griseoincarnatus SSPJ4 emerged as the most promising isolate, demonstrating significantly higher enzyme activities. S. griseoincarnatus SSPJ4 was subjected to whole-genome sequencing using the GALAXY tool for assembly and annotation, with an assembled genome of 7.3 Mb, 72.38% G+C content, and 6678 genes. Biosynthetic gene clusters (23) were identified using antiSMASH, and 226 proteins were annotated with CAZy domains, of which 121 belonged to the glycoside hydrolase (GH) family. Supplementation of Celluclast® with the crude enzyme preparation from S. griseoincarnatus SSPJ4 enhanced the saccharification of alkali-pretreated paddy straw, resulting in a 1.55-fold higher sugar yield compared with Celluclast® alone. Conclusion: The findings demonstrate the considerable potential of S. griseoincarnatus SSPJ4 as a source of accessory lignocellulolytic enzymes for improving biomass saccharification. Its enzyme repertoire could be exploited to develop cost-effective and efficient enzymatic formulations for second-generation bioethanol production. https://ijm.tums.ac.ir/index.php/ijm/article/view/5702 https://ijm.tums.ac.ir/index.php/ijm/article/download/5702/1916