Approximately 10 billion tons of dry plant matter are produced globally each year. Over half of it is composed of cellulose or hemicellulose. These polysaccharides form the largest reservoir of organic carbon on Earth [1], [2]. For instance, agricultural wastes, byproducts or biomasses rich in cellulosic materials, remain largely underutilized. This lignocellulosic biomass is a sustainable resource for producing biotechnological value-added products, such as textiles, chemicals, and biofuel [3]. The conversion of biomass to biofuel is facilitated by cellulolytic enzymes [4], [5], [6]. However, the production of these cellulolytic enzymes is costly and novel efficient methods need to be developed.

Enzymes can be produced efficiently utilizing fungi, due to their ease cultivation and high yield of enzymes produced [7].Bairagi 2016). In addition to the fungal genus Trichoderma [6], [8], [9], [10],high production of extracellular cellulases have been reported for the genera Aspergillus [11], Penicillium [12], [13], and Humicola [14].

In biotechnology, improving fungal strains is crucial to improve the efficiency of the technology. Research improving the efficiency of cellulase production is one priority in biomass studies. Several strategies can help reduce the cost of cellulase production, including genetic improvement techniques [1], [15], [16]. Industrial strains are typically developed through traditional (non-GMO) improvement methods, including random screening and selection. Recombinant DNA techniques are playing a crucial role in the generation of strains for industrial use. Recent advancements have enabled the enhancement of microbial strains in many industrial areas [12]. The development of novel hybrid strains offers a considerable potential to improve the efficiency of biofuel production from biomasses [17].

Protoplast fusion is a genetic modification tool serving a bypass from the traditional cell mating. It opens new avenues for developing microorganisms with novel properties [16], [18], [19]. Protoplast fusion is a whole-genome mixing technique where the outcome is fusant strains with combined traits from both parents. This approach provides tools for increasing gene dosage and expression through strong promoters, deleting undesirable genes from fungal genomes, manipulating metabolic pathways, and developing fungal strains for heterologous protein production. Cellulase genes and their regulatory systems may differ across species or genera of cellulase-secreting microorganisms. These genes can be recombined through protoplast fusion, followed by advanced screening techniques to select superior hybrid strains [20]

In this investigation, fungal strains with high cellulolytic activity are isolated from decomposing wood. The fungal isolates with high cellulolytic activity is identified molecularly and protoplast fusion is performed with the aim to enhance the extracellular cellulolytic enzyme activity of the novel fusants. The DNA banding patterns of hyper-cellulolytic fusants and their parental strains are compared using four RAPD and three ISSR primers. Finally, the study will concentrate and purify the highest cellulolytic hybrid fusant strain using ultrafiltration, SDS-PAGE, and zymogram assays.