Screening and Characterization of Lignocellulolytic Microbes from the Tillage Ecosystems for Sustainable Valorization of Agro-waste

Screening and Characterization of Lignocellulolytic Microbes from the Tillage Ecosystems for Sustainable Valorization of Agro-waste | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Screening and Characterization of Lignocellulolytic Microbes from the Tillage Ecosystems for Sustainable Valorization of Agro-waste Ammara Fatima, Adnan Zahid, Sajid Ali, Waheed Anwar, Amina Arshad, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7533898/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Burning of crop residues contributes significantly to air pollution, increases black carbon emissions, and accelerates climate change. Sustainable alternatives involve returning residues to the soil and applying lignocellulolytic microorganisms to speed up their breakdown, thereby supporting eco-friendly farming systems. This study focused on isolation and screening of the lignocellulolytic microbes particularly bacteria and fungi from tillage management scenarios as a viable alternative to crop residue burning. A total of 10 fungal and 5 bacterial strains were isolated in the form of pure colonies and their lignocellulolytic potential was screened by qualitative and quantitative screening. Primarily, the lignocellulolytic degradation was evaluated by using carboxymethylcellulose (CMC) and Tannic Acid (TA) agar media, on the basis of appearance of zone formation. Microbial strains FT4A, FT1B, BT4, BT2, FT2A and FT4A were showed the highest length of cellulolytic and lignolytic zone formation. Secondly, they were quantitatively screened by standard protocols of CMCase, FPase and Laccase enzyme assay techniques. FT1B and BT4 have the highest cellulolytic values 11.6 U/mL and 6.7 U/mL in CMCase assay. In FPase assay, FT2B and BT3 have the highest cellulolytic values 7.7U/mL and 0.14 U/mL. FT4A had highest lignolytic potential 0.14U/mL in Laccase assay and many isolates had least activity. The potential colonies which had significant results were identified as Aspergillus flavus, EmmonsiaPasteurina, Aspergillus niger , Penicillium sp, Monococcusechinophorus, Bacillus subtillus, Streptococcus sanguis and Pseudomonas flourescens . These species will be very useful if they apply in-situ crop residue degradation which ultimately improve the air quality, reduce environmental pollution and conservation of microbial biodiversity. Lignocellulose degradation Cellulases Lignin peroxidase agro-waste Bacteria Fungi Waste valorization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Recent challenges to food security, driven by climate change, limited arable land, and soil degradation, are exacerbated by intensive agricultural practices and the widespread use of agrochemicals, which have boosted global production (Zarezadeh et al., 2025 ). However, these practices have led to the accumulation of significant organic waste, causing environmental pollution, soil health deterioration, and increased public health risks. Traditional waste management methods, including burning, landfilling, and chemical degradation, are inefficient, energy-intensive, and harmful to the environment (Popp et al., 2021 ). The nexus between waste accumulation and deteriorating soil health underscore the urgency in addressing the rising threat to global food security (Jiang & Zhou, 2023 ), aligning with the United Nations' Sustainable Development Goals (Nations, 2018 ). With the global population projected to reach 10 billion by 2050, the need for sustainable agricultural systems is urgent, particularly in arid regions that support a third of the world’s population. As a result, there has been a shift towards sustainable, eco-friendly agri-technologies that offer value-added solutions for organic waste management, aiming to address pollution and support long-term environmental health (Dar et al., 2024 ). Crop residues(CRs) which remain after harvest, are significant renewable resources and by-products of agriculture production. Being rich in macro- and micronutrients, CRs are pivotal for plant growth. Further, CRs account for approximately 40% of the total dry biomass, they are crucial for maintaining the stability of agricultural ecosystems (Sharma et al., 2022 ). Amendment of CRs enhances organic matter content, boosts crop productivity, improves soil nutrients, and reduces essential nutrient depletion. These CRs are majorly composed of lignocellulose produced in tremendous quantities worldwide (Singh et al., 2022 ). Recent estimates suggest that global lignocellulose biomass production reaches approximately 181.5 billion tons annually, yet only 8.2 billion tons are currently utilized across various applications (Mujtaba et al., 2023 ). The lignocellulosic biomassis composed of proteins, salt, minerals, ash, lignin (an aromatic polymer), pectin (a heteropolysaccharide), carbohydrates (such as cellulose and hemicellulose), and other compounds, the compositions and proportions of lignocellulose compounds vary among different plants (Dar et al., 2022 ; Dar et al., 2024 ). Plant cell wall layers contain lignin, cellulose, and hemicellulose as the middle lamella of a plant's cell wall contains the highest concentration of lignin, and the S2-layer of the secondary wall is particularly rich in cellulose (Manavalan et al., 2015 ). Several organisms are known to degrade and utilize lignocellulosic biomass known as natural biomass utilization systems (Dar et al., 2021 ). In nature, the degradation of lignocellulosic biomass occurs through the coordinated action of various microorganisms, predominantly fungi and bacteria, which secrete a range of cellulolytic and hemicellulolytic enzymes under both aerobic and anaerobic conditions (Ali et al., 2024 ; Dar et al., 2015 ). Among these bio-resources bacteria and fungi are preferred biocatalysts due to their ease of cultivation, short growth periods and easy to bioengineer for enhanced activities (Xie et al., 2023 ). During the last decade, cellulase-producing bacteria have been isolated from diverse environments, including composting plants, decaying plant material from forestry or agricultural waste, ruminant feces, soils, organic matter, and the gastrointestinal tracts of animals (Sun et al., 2023 ). Although numerous cellulolytic bacteria have been documented across diverse ecological niches, the efficient and economically viable bioconversion of lignocellulosic biomass into commodity products remains a significant challenge (Adegboye et al., 2021 ). Consequently, there is an urgent need for bio-prospection aimed at identifying more effective bacterial strains to enhance the bioconversion efficiency into value-added products. Isolating and purifying native bacteria and fungus from agricultural soils and decaying organic matter was the goal of this investigation. It used both primary and secondary screening techniques to assess their enzymatic activity and biodegradation potential. Finding viable microbial strains that could improve in-situ agricultural residue breakdown for quick, economical, and ecologically friendly composting methods was the aim. To reduce the negative effects of in situ burning and to maximize the use of various crop residues, an affordable and environmentally friendly residue management approach should be adopted (Shukla et al., 2014 ). An eco-friendly management strategy that uses lignocellulose-degrading microbes to hasten the degradation of CR is an alternative to avoiding environmental pollution (Garg, 2017 ). An effective method for accelerating the potential of lignocellulose degradation in agricultural residue is the inoculation of ligno-cellulolytic microorganisms. Numerous microbes including bacteria and fungi can produce enzymes for the breakdown of lignin, cellulose and hemicelluloses (Manavalan et al., 2015 ). It has become necessary to isolate and inoculate rice straw-degrading microbes to accelerate the decomposition of rice straw prior to wheat seeding. This will enable the use of standard zero-till planters and minimize the negative impacts on wheat growth. Even though residue-degrading bacteria and fungi are already present in soil, the amount of these fungi varies substantially between fields and locations based on environmental, edaphic, and management factors (Choudhary et al., 2015 ). According to our hypothesis, field inoculation will increase the population density of fast-degrading autochthonous fungi once they have been isolated and grown in fields under Rice-Wheat (RW) systems. MATERIALS AND METHODS Soil Sample Collection: Soil samples were collected from long term residue management and conservation tillage experiment at Rice Research Institute (31°72’ N, 74°28’ E, Slope 1.8%), at Kala Shah Kaku (RRI, KSK), Shiekhupura, and Punjab, Pakistan. The five different tillage scenarios were based on cropping management, combined tillage and crop leftover management practices. In scenario-I; conventional tillage practices were used, scenario-II was led to no burning and ploughing, scenario-III was zero-tillage and non-puddled rice, scenario-IV was zero-tillage with puddled rice and scenario-V had conservation tillage (Zahid et al., 2020 ). Soil samples were packed in the sterile polythene bags, labeled as T1, T2, T3, T4 and T5, respectively as per the scenario. After sample collections, the soil samples were transferred to the lab and maintained at 4 ℃. Isolation and Purification of Microbes: Microbes were isolated from these soil samples to screen their lignocellulolytic potential. Serial dilution technique was used to isolate the fungal and bacterial strains from each five-soil samplevizT1, T2, T3, T4 and T5. After 10⁻⁵ dilutions, 1mL of last diluted soil sample was poured drop wise on already prepared PDA media plates for fungi and LBA media plates for Bacteria. PDA media plates were incubated at 28˚C for maximum 7–10 days and LBA media plates were incubated at 37˚C for 48 h. The microbial growth was observed within these days by following previous methods (NAYAK et al., 2017; Shinde et al., 2022 ). On the basis of morphology, 2 different fungal colonies and 1 bacterial colony was isolated from each soil sample plate and transferred antiseptically on already prepared pertinent media plates to obtain pure culture. All plates were accurately labeled with different code name according to soil sample to avoid any uncertainty; FT1A, FT1B, FT2A, FT2B, FT3A, FT3B, FT4A, FT4B, FT5A and FT5B for fungal colonies and BT1, BT2, BT3, BT4 and BT5 for bacterial colonies. Purified colonies without any contamination were preserved in refrigerator at 4˚C for screening and identification (NAYAK et al., 2017; Shinde et al., 2022 ). Molecular Identification of Microbes: For bacterial and fungal Identification DNA extraction was conducted using the cetyltrimethylammonium bromide (CTAB) method (Schenk et al. 2023 ). For fungal species, ITS primer were used. PCR amplifications for ITS, initial denaturation for 5 min at 95°C, followed by 35 cycles of denaturation at 94°C (30 s), annealing temperature at 56°C or 59°C (30 s), and extension at 72°C for 1 min. The amplification was accomplished with one additional step of final extension at 72°C for 10 min.(Asis et al. 2021 ). The PCR conditions for Bacteria were 3 min at 95°C for 32 cycles, 1 min at 94°C, 1 min at 56°C, 2 min for 72°C, and 10 min for 72°C with 4°C intervals. After sequencing, the obtained sequences were blasted using the online tool NCBI Blast (Chukwuma et al. 2023 ). Phylogenetic tree was made of different fungal and bacterial isolates by using MEGA X software. Phylogenetic analysis of Microbes. The bacterial isolate 16S regions and the fungal isolates' ITS (Internal Transcribed Spacer) regions, as well as closely related reference sequences that were obtained from the NCBI GenBank database ( http://ncbi.nih.gov ), were used to create a phylogenetic tree (Asis et al. 2021 ). To guarantee the clustering pattern's dependability, the Neighbor-Joining technique was used in MEGA X for the analysis, which included 1000 bootstrap replications. MEGA X software was used to construct phylogenetic tree. In-vitro Primary Screening of Potential Lignocellulolytic Microbes: Primary Screening also known as qualitative screening was employed to determine the potential of the isolated microbes. To accomplish this, qualitative agar plate method was used for primary screening of lignocellulolytic fungi and bacteria. The appearance of the hydrolysis zone formed after the growth of the microorganisms was deemed as positive for lignocellulose degradation (Gahfif et al., 2020 ). Cellulolytic Degradation: The cellulolytic or endoglucanase activity was tested by using the CMC agar media. The media was prepared with the following constituent’s g/500mL; 0.5g yeast extract, 13g carboxymethyl cellulose, 1.5g agar and trace elements 0.5g MgSO 4 .7H 2 O, 0.1g KCL and 0.5g NH 4 H 2 PO 4 (Hankin & Anagnostakis, 1977 ) with modifications. Purified colonies of both microbes were inoculated on the CMC agar media plates (Choudhary et al., 2016 ; Kausar et al., 2010 ). Then, after their incubation and proper growth 1% Congo red dye solution was flooded in the media plates for 20 min. Subsequently, the plates were drained and washed with 1M NaCl solution for 20 min. A CMC clearance zone was appeared around fungal and bacterial colonies indicating cellulolytic potential. This clear zone was measured by the following formula to calculate the Enzymatic Index (EI) (Marđetko et al., 2021 ; Namnuch et al., 2021 ): $$\:Enzymatic\:Index\:\left(EI\right)=Diameter\:of\:hydrolysis\:zone/Diameter\:of\:colony$$ Lignolytic Degradation: Oxidative test was used to check the lignin degradation of fungi. Phenol oxidase was considered as a key element of lignin degradation. Tannic Acid agar media was used to screen the lignin degradation capacity of fungi by verifying the presence of polyphenol oxidase. Tannic Acid agar media was prepared by adding 7.5g malt extract agar, 10g agar and 2.5g Tannic acid under the sterile conditions and protective method (Dabhi et al., 2017 ; Thormann et al., 2002 ). Then, purified fungal colonies were inoculated on these Tannic Acid agar media plates and incubated at 28˚C in dark place for 7-10days. The brownish or greenish color zone formation around the fungal colonies was the result of polyphenol oxidase degradation (Choudhary et al., 2016 ; Kausar et al., 2010 ). In-vitro Secondary Screening of Potential Lignocellulolytic Microbes: Secondary screening is the quantitative estimation of lignocellulolytic enzymes of fungal and bacterial strains. Basal broth media was used for the production of these enzymes. Under submerged fermentation, broth media was prepared by using the following constituents; CMC, wheat residue, KH 2 PO 4 , CaCl 2 .2H 2 O, Urea, MgSO 4 .7H 2 O, (NH 4 )2SO 4 , Peptone, yeast Extract, Tween-80, FeSO.7H 2 O, MnSO 4 .H 2 O and ZnSO 4 .7H 2 O. All these ingredients with concentration g/1100 given in Table 1 were added in to distilled water (Awadalla et al., 2017 ; Cyrus & Juwon, 2015 ). Table 1 Concentrations of the components involved in the preparation of Broth Media. Sr. No Ingredients Concentration (g/1100) 1. Carboxymethyl Cellulose 6g 2. wheat residue 5g 3. KH 2 PO 4 2.2g 4. CaCl 2 .2H 2 O 0.33g 5. Urea 0.33g 6. MgSO 4 .7H 2 O 0.33g 7. (NH 4 )2SO 4 1.54g 8. Peptone 0.275g 9. yeast Extract 0.11g 10. Tween-80 1.1mL 11. FeSO 4 .7H 2 O 0.0055g 12. MnSO 4 .H 2 O 0.00176g 13. ZnSO 4 .7H 2 O 0.00154g After the inoculation of both microbes, flasks with fungal inoculation were placed in to shaking incubator at 28˚C and 160 rpm for 7 days and with bacterial inoculation were placed in to shaking incubator at 37˚C and 160 rpm for 24 h. Then, the incubated broth was filtered and centrifuged at 10,000 rpm for 10 min at 4˚C. The clear supernatant was accumulated and stored at 4˚C. These crude enzymes were further used for the quantitative lignocellulolytic analysis of these enzymes by CMCase, FPase and Lacase assay techniques (Choudhary et al., 2016 ; Namnuch et al., 2021 ). CMCase and FPase Assay: CMCase and Filter Paperase assay technique were used for Endo-β-1,4-Glucanase test. The reported procedure of (Ghose, 1987 ; Miller, 1959 ) was followed by using 2% CMC and filter paper strips of size 1cm x 6cm as a substrate respectively. Sodium citrate buffer and substrate was thoroughly mixed in test tubes and placed in to incubator at 50˚C for 30 minutes. DNS (3 5- dinitrosalicylic Acid) (Miller, 1959 ; Sivaramanan, 2014 ) added to consummate the enzymatic reaction and for measuring the reducing sugar. UV spectrophotometer was used to measure the optical absorbance of reducing sugar of each sample at 540nm against spectro zero. The readings of optical absorbance of main samples were subtracted from their enzyme blanks and spectro zero. These readings were compared and plotted with glucose standard curve. Under the assay conditions, the amount of enzyme that was required to release 1 µmole of reducing sugar per min was one unit of enzyme activity (Awadalla et al., 2017 ; Cyrus & Juwon, 2015 ). Cellulase Activity was calculated as per formula; Cellulase activity= \(\:{\mu\:}\text{m}\text{o}\text{l}\text{e}\text{s}\times\:{\text{V}}_{\text{t}}/{\text{V}}_{\text{s}}\times\:\text{t}\) (Adney & Baker, 2008 ) Laccase Assay: This assay technique was based on the oxidation of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) method used to quantify the lignolytic potential of fungal enzymes. The reaction mixture was prepared by adding 10mM ABTS and the absorbance of the mixture was measured by using UV-spectrophotometer at 420nm. (NAYAK et al., 2017; Zhang et al., 2021 ) The amount of enzyme that was needed to oxidize 1 µmole of ABTS per min with molar absorbance 360000 M ⁻ 1 cm⁻ 1 is the one unit of enzyme activity (Marđetko et al., 2021 ). Laccase enzyme was measured by the following formula (Baltierra-Trejo et al., 2015 ): $$\:\varvec{E}\varvec{n}\varvec{z}\varvec{y}\varvec{m}\varvec{e}\:\varvec{A}\varvec{c}\varvec{t}\varvec{i}\varvec{v}\varvec{i}\varvec{t}\varvec{y}\frac{1\varvec{U}}{\varvec{m}\varvec{l}}\:=\frac{\varDelta\:\varvec{A}\varvec{b}\varvec{s}\times\:\varvec{V}\varvec{t}}{\in\:\:\times\:\varvec{I}\varvec{n}\varvec{c}\varvec{u}\varvec{b}\varvec{a}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\:\varvec{t}\varvec{i}\varvec{m}\varvec{e}\:\times\:\varvec{V}\varvec{e}\:\times\:\varvec{p}\varvec{a}\varvec{t}\varvec{h}\:\varvec{l}\varvec{e}\varvec{n}\varvec{g}\varvec{t}\varvec{h}}.$$ Identification of Lignocellulolytic Microbes: Fresh fungal and bacterial colonies were firstly identified macroscopically by their traits of size, shape, color and growth. Then, slides of all cultures were prepared to analysed them microscopically under Microscope Nikon Eclips E200 to see hyphal, spores and conidial shapes and different spherical, spiral and other bacterial shapes by 10X and 40X lenses and identified by various keys (John I Pitt & Ailsa Diane Hocking, 2009). Statistical analysis: Randomized Control design was used for all experiments with three replications. The recorded data was statistically analyzed by ANOVA using (Statistics 8.1) and Least Significant Difference (LSD) was applied to find the significance between the different microbial colonies. RESULTS Isolation and Purification of Fungi: As total of 10 fungal isolates and 5 bacterial isolates were isolated on the basis of different morphology, 3 isolates FT1A, FT1B and BT1 from the Scenario-I soil sample that was labeled as T1, 3 isolates FT2A, FT2B and BT2 from the Scenario-II (T2), 3 isolates FT3A, FT3B and BT3 from Scenario-III (T3), 3 isolates from Scenario-IV (T4) and 3 Isolates from Scenario-V soil sample (T5). These isolates were selected on their different dominant characteristics of color and shape given in the Table. 2 and manifested in Fig. 1 . Table 2 Dominant characters of microbial colonies for their selection and purification. Sr. No Strains Color of Colonies Shape of Colonies Texture of Colonies 1. FT1A White to Greyish 1 Round Cottony 2. FT1B Green Rounds Spore or granular 3. FT2A Pure white Round Thick Hairy 4. FT2B Light Green & white pink corners Wrinkle Round Hairy & spore 5. FT3A Light Pink white Irregular Weak Hairy 6. FT3B Dark Greenish Wrinkle Round velvet 7. FT4A Pure Black Round Spore or granular 8. FT4B Green & white Round Hairy 9. FT5A Light Green, white & pink Wrinkle Round Hairy and spore 10. FT5B Greyish Wrinkle velvety 11. BT1 Creamy White Comma Soft Creamy 12. BT2 Greyish White Bacilli Soft Creamy 13. BT3 Light Brown Cocci Soft Creamy 14. BT4 Yellow Cylindrical Soft Creamy 15. BT5 White Club Rod Soft Creamy Phylogenetic Tree of Fungal Isolates Based on ITS Sequences The phylogenetic tree was constructed based on the alignment of ITS (internal transcribed region) gene sequences to determine the evolutionary relationships among the isolated fungal strains. Aspergillus niger, Aspergillus flavus, Penicillium sp., and Emmonsia sp. were among the closely related reference strains that the isolates clustered with, according to the analysis. Accurate identification and evolutionary relation were confirmed by this tree, which shows genetic similarity between the isolates and their corresponding reference sequences. The resulted fungal isolates tree clearly showed that the isolates group within distinct clades corresponding to their respective genera and species, demonstrating high similarity with known reference strains such as Aspergillus niger , and Penicillium spp. The close clustering of fungal isolates with authenticated reference sequences confirms their molecular identity and evolutionary placement. This analysis not only validates morphological identification but also provides insights into the genetic diversity and relatedness of the studied fungal community. The presence of Emmonsia sp. as a separate branch reflects their genetic divergence from Aspergillus and Penicillium. Overall, the phylogenetic analysis effectively differentiates between species and underscores both inter- and intra-species diversity among the sampled fungi, supporting the taxonomic classification and genetic variations (Fig. 2 ). Phylogenetic Tree of Bacterial Isolates Based on 16S rRNA Sequences The phylogenetic tree was constructed based on the alignment of 16S rRNA gene sequences to determine the evolutionary relationships among the isolated bacterial strains. The analysis revealed clustering of the isolates with closely related reference strains, including Streptococcus sanguis , Pseudomonas fluorescence , and Bacillus subtilis . The 16S rRNA gene sequence analysis confirmed the molecular identity of the bacterial isolates obtained in this study. The phylogenetic tree showed that Bacillus subtilis BT2 clustered closely with reference strains of Bacillus subtilis , indicating a high degree of similarity and confirming its identification. Similarly, Pseudomonas fluorescens BT4 grouped with authentic strains of Pseudomonas fluorescens , demonstrating strong evolutionary relatedness. Interestingly, Bacillus subtilis BT2 showed close alignment with Streptococcus sanguis BT3, suggesting genetic similarity or possible horizontal gene transfer events. Overall, the clustering pattern verified that the isolates belonged to well-established bacterial taxa and highlighted their evolutionary relationships with closely related species deposited in the NCBI database (Fig. 3 ). Primary Screening of Lignocellulolytic Microbes: The Purified isolates were then screened on solid plate media to check their cellulose and lignin degradation capability. The zones were appeared around colonies that showed their ligocellulolytic potential. These zones were measured by a cm scale to compare the colonies potential. Cellulolytic Degradation: The clear zone or yellow opaque color zone was formed around the purified colonies on CMC agar media by applying congo red dye solution. This clear zone was visualized the hydrolysis of cellulose by both fungal and bacterial enzymes. All the colonies were showed the zone formation with the varying capability of cellulose breakdown. They were showed their cellulolytic activity in the range 0.1cm to 1.6cm. It was estimated by measuring the one-side length (Radius) of the zone formation. Fungal isolates FT4A, FT1A, FT1B and FT3A were showed the highest length of cellulolytic zone formation that was 1.5 cm, 1.1 cm, 1.2 cm and 1 cm respectively. Bacterial isolates BT2 and BT4 showed the highest length of cellulolytic zone formation that was 1.5 cm and 1 cm respectively. The length of other isolates was mentioned in the Table.3.2 and visualized in Fig. 4 . Lignolytic Degradation: The greenish or brownish color zone was formed around the purified fungal colonies on Tannic Acid agar media. This zone formation was the hydrolysis of polyphenol oxidase by the fungal enzymes that demonstrated the lignin activity. All the fungal colonies were not showed the zone formation; only few isolates had varying ability of lignin breakdown. They were showed their lignolytic activity in the range 0.1cm to 1cm. It was estimated by measuring the one-side length (Radius) of the zone formation. Fungal isolates FT2A, FT4A and FT5B were showed the highest length of lignolytic zone formation and their values were 1cm, 0.9cm and 0.8cm respectively. The length of zone formation of other isolates was mentioned in the Table.3 also showed in Fig. 4 . Table.3. Qualitative Screening of Potential Lignocellulolytic Microbes on Agar Media Plates. Sr. No Microbial Strains Source of the strains Lignocellulolytic Activity Check Enzymatic Index of Lignocellulolytic Zone Formation (cm) Cellulose Degradation on CMC agar (Radius) Lignin Degradation on Tannic Acid agar (Radius). 1. FT1A Soil from S1 +, - 1.1 ± 0.02 - 2. FT1B Soil from S1 +, - 1.2 ± 0.03 0.1 ± 0.002 3. FT2A Soil from S2 +,+ 0.5 ± 0.01 1 ± 0.02 4. FT2B Soil from S2 +, - 0.1 ± 0.002 - 5. FT3A Soil from S3 +, - 1 ± 0.02 - 6. FT3B Soil from S3 +, - 0.7 ± 0.01 0.16 ± 0.004 7. FT4A Soil from S4 +, - 1.5 ± 0.03 0.9 ± 0.23 8. FT4B Soil from S4 +, - 0.2 ± 0.005 - 9. FT5A Soil from S5 +, + 0.6 ± 0.01 - 10. FT5B Soil from S5 +, - 0.5 ± 0.01 0.8 ± 0.20 11. BT1 Soil S1 + 0.5 ± 0.01 - 12. BT2 Soil S2 + 1 ± 0.02 - 13. BT3 Soil S3 + 0.5 ± 0.01 - 14. BT4 Soil S4 + 1.25 ± 0.03 - 15. BT5 Soil S5 + 0.5 ± 0.01 - Secondary Screening of Lignocellulolytic Microbes: Quantitative Screening of all fungal enzymes was measured on CMC liquid broth media amended with wheat straw. CMCase, FPase and Lacase assays were performed for the quantification of enzyme activity of Lignocellulolytic fungi. CMCase Assay: The 1U/mL/min cellulolytic enzyme activity of all microbial enzymes was measured by using slope value and enzyme activity calculations. All the enzymes had cellulolytic activities with different concentrations. Cellulolytic activity of CMCase assay was ranged from 1.1 U/mL to 11.6 U/mL as given in Table. 4. Fungal strains FT1A, FT1B and FT4A have the highest cellulolytic values 5.8 U/mL, 11.6 U/mL and 6.7 U/mL in CMCase assay. Bacterial strains BT4 and BT2 have the highest cellulolytic values 6.75 U/mL and 2.19 U/mL in CMCase assay. Figure 5 and Graphs (a & b) represented the significant difference of both bacterial and fungal hydrolytic enzyme activities. FPase Assay: The 1U/mL/min cellulolytic enzyme activity of all enzymes in FPase was also measured by using slope value and enzyme activity calculation. In FPase all the enzymes also had cellulolytic activities with different concentrations. Cellulolytic activity of FPase assay was ranged from 0.04 U/mL to 7.7 U/mL as given in Table.4. Fungal strains FT2B and FT1B have the highest cellulolytic values 7.7U/mL and 6.9 U/mL in FPase assay. Bacterial strains BT5 0.62U/mL and BT2 0.14U/mL have the highest cellulolytic values. FPase assay values were different from CMCase assay. Figure 5 . and Graph (c & d) showed the significant values of FPase assay. Laccase Assay: Laccase assay was used to measure the lignin activity of all fungal enzymes. Enzyme activity 1U/mL/ min was calculated by the oxidation of substrate ABTS. Fungal enzymes showed very less lacase activities. The range of activity was from 0.089 U/mL to 1.14U/mL. The highest value of lacase was showed by FT4A and FT5A had lowest value about negligible. Table. 4 and Fig. 5 graphs displayed the significant ranges of quantitative lignolytic activity of fungi. Table.4 Quantitative Screening of Potential Lignocellulolytic Microbes by submerged fermentation. Sr.No Microbial Strains CMCase Activity (1U/ml/min) FPase Activity (1U/ml/min) Laccase Activity (1U/ml/min) 1. FT1A 5.8 ± 0.14 4.9 ± 0.12 0.12 ± 0.003 2. FT1B 11.6 ± 0.29 6.9 ± 0.17 0.11 ± 0.002 3. FT2A 4.5 ± 0.11 4.73 ± 0.15 0.13 ± 0.003 4. FT2B 2.72 ± 0.06 7.7 ± 0.19 0.012 ± 0.0003 5. FT3A 3.5 ± 0.08 5.5 ± 0.13 0.095 ± 0.002 6. FT3B 3.954 ± 0.09 4 ± 0.10 0.087 ± 0.002 7. FT4A 6.7 ± 0.16 6.65 ± 0.16 0.14 ± 0.003 8. FT4B 4.05 ± 0.10 6.5 ± 0.16 0.12 ± 0.003 9. FT5A 2.2 ± 0.05 7.2 ± 0.18 0.089 ± 0.002 10. FT5B 1.5 ± 0.03 6.7 ± 0.16 0.13 ± 0.003 11. BT1 1.95 ± 0.04 0.04 ± 0.001 - 12. BT2 2.19 ± 0.05 0.10 ± 0.002 - 13. BT3 1.74 ± 0.04 0.14 ± 0.003 - 14. BT4 6.75 ± 0.2 0.62 ± 0.01 - 15. BT5 1.59 ± 0.04 0.12 ± 0.003 - Identification of the isolated Lignocellulolytic Microorganisms: The macroscopic characteristics were observed by their color, size, shape, texture and growth. The microscopic traits of fungi were observed by the shape of Hyphae (septate and non-septate) and shape of Conidia (regular, irregular, round and rough e.t.c) and bacteria was observed by their shapes: spherical (cocci), rod (bacilli), spiral (spirilla), comma (vibrios) or corkscrew (spirochaetes). These differentiating attributes were specified in Table. 5 and be revealed in Fig. 6 . Table 5 Microscopic characteristics of Lignocellulolytic Microbes. Sr.No Microbial Strains Nature of Hyphae Conidia Shape Form Margin 1. FT1A Filamentous - - - 2. FT1B Septate Rough - - 3. FT2A Septate Round - - 4. FT2B Septate Spherical - - 5. FT3A Septate Globose - - 6. FT3B Septate Irregular - - 7. FT4A Septate Globose - - 8. FT4B Septate Rough - - 9. FT5A - Irregular - - 10. FT5B Septate Spherical - - 11. BT1 - - Irregular small Undulate 12. BT2 - - Irregular small Flat 13. BT3 - - Regular small Lobate 14. BT4 - - Regular large Smooth 15. BT5 - - Irregular small Lobate Macroscopic and microscopic identification of potential lignocellulolytic strains revealed; FT1B as Aspergillus flavus , FT2A as EmmonsiaPasteurina , FT4A as Aspergillus niger , FT5B as Penicillium sp. , BT1 as Monococcusechinophorus , BT2 as Bacillus subtillus , BT3 as Streptococcus sanguis and BT4 as Pseudomonas flourescens (John I Pitt & Ailsa D Hocking, 2009). DISCUSSION Lignocellulosic waste is the most abundant biorenewable biomass on earth, and its hydrolysis releases highly valued reducing sugars. However, the presence of lignin in the biopolymeric structure makes it highly resistant to solubilization thereby hindering the hydrolysis of cellulose and hemicellulose. Microorganisms are known for their potential complex enzymes that play a dominant role in lignocellulose conversion (Zhang et al., 2021 ). Plant cell walls are degraded and assimilated by microorganisms in a complex process that involves the cooperative action of many different organisms and the enzymes outside of cells. In plants, the cell wall primarily complex polysaccharides (cellulose, Hemicellulose and pectin) which together make up a dense substance, coated phenolic polymer with multiple uses (M'barek et al., 2019 ). The lignocellulosic biomass is broken down by cellulases to produce fermentable sugars. Natural lignocellulose must be saccharified enzymatically, and when working with lignocellulosic substrates, the use of cellulose-degrading enzymes is crucial (Donohoe & Resch, 2015 ). Biomass burning after crop harvesting is the most opted choice for farmers in order to manage residues. Though it is an easier option but it is not eco-friendly and has negative effects on the environment such as release of greenhouse gases and fine particulate matter. An alluring alternate strategy was used of Lignocellulolytic microbes. So, present study is aimed to come up with a viable alternative to crop residue burning as a way to attain sustainable alternative to protect the environment and explore the ability of potent microbes to selectively delignify. Total 15 microbial isolates were isolated on the basis of different morphology from cropping management scenarios. The purified isolates were then screened on solid plate media to check their cellulose degradation capability. The clear zone or yellow opaque color zone was formed around the purified colonies on CMC agar media. All the colonies were showed the zone formation with the varying ability of cellulose breakdown. The fungal colonies showed their cellulolytic activity in the range 0.1cm to 1.6cm and bacterial colonies showed their activity in the range 0.5cm to 1.25cm. Several researchers (Choudhary et al., 2016 ; Keerthana et al., 2019 ) reported similar findings. Cellulase is an enzyme that the cellulolytic microorganisms make that can break down cellulose into monomeric or dimeric forms that are soluble in water and can be used as a carbon or food source for colony growth (Romsaiyud et al., 2009 ). Five isolated microbial cultures (IMCs) demonstrated the development of clear zones around the inoculation site, which are easily visible after staining with Congo red dye. The seven active microbial cultures had zone formations with diameters ranging from 1.1 to 2.9 cm. The sample IMC 18's maximum zone was 2.9 cm, whereas the sample IMC 4's maximum zone was 2.1 cm. IMC 11 showed a moderate clear zone measuring 1.9 cm in diameter, with zone diameters of 1.2 cm for IMCs 6 and 17, and 1.1 cm for IMCs 1 and 20. The remaining 14 cultures had no discernible distinct zone (Shinde et al., 2022 ). For lignolytic screening on agar media, fungal cultures were grown on Tannic acid agar media. All the fungal cultures were not showed the zone formation; only few isolates had varying ability of lignin breakdown. They were showed their lignolytic activity in the range 0.1cm to 1cm. On tannic acid media, isolate F44 developed a considerably (p 0.05) larger dark brown zone (84.71%). The tannic acid medium developed a dark brown zone, which demonstrated the fungal isolate's polyphenol oxidase (PPO) activity. In order to create dark brown complexes, which are essential for the breakdown of the phenolic molecule in lignin, polyphenol oxidase, a combination of monophenol oxidase and catechol oxidase, catalyzed the reaction between polyphenol and molecular oxygen. During the biodegradation of lignocellulosic materials, fungi have been demonstrated to create ligninolytic enzymes (Rodrigues et al., 2008 ). (Dinis et al., 2009 ) also reported on the activities of lignin peroxidases, manganese peroxidases, and laccase during solid state fermentation of lignocellulosic materials. Quantitative screening of all microbial hydrolytic enzymes was measured on CMC liquid broth media amended with wheat straw. CMCase, FPase and Lacase assays were performed for the quantification of enzyme activity of Lignocellulolytic microbes. Fungal strains FT1A, FT1B and FT4A and Bacterial strains BT4 and BT2 have the highest cellulolytic values 5.8 U/ml, 11.6 U/ml, 6.7 U/ml, 6.75 U/ml and 2.19 U/ml respectively in CMCase assay. FT2B, FT1B, BT5 and BT2 have the highest cellulolytic values 7.7U/ml, 6.9 U/ml, 0.62U/ml and 0.14U/ml respectively in FPase assay. FT4A had highest lignolytic potential 0.14U/ml in Lacase assay and many isolates had least activity. The highest CMCase activity of IMC 18 was (0.26 IU/ml), followed by IMC 4 (0.21) and IMC 11 (0.16). IMC1 had the least amount of activity (0.05) and several cultures, such as IMC 6 (0.07), IMC 20 with 0.08 and 17 (0.09) were found to possess a moderate CMCase activity. When FPase utilization of the Whatman No. 1 filter to quantify activities IMC 18 once more shown the highest activity of IMC 4 was second highest at 0.14 IU/ml 0.11 MU/ml. IMC 6 displayed a low FPase activity (0.008), although IMC 1, 11, 17, and 20 did not placed in the middle. FPase activity was overall ranged between 0.02 and 0.14 for each of the seven colonies (Shinde et al., 2022 ). Then, they were firstly identified macroscopically by their traits of size, shape, color and growth and microscopically by their traits of shape of hyphae and conidia shape. The potential Lignocellulolytic fungal colonies were identified FT1B as Aspergillus flavus , FT2A as EmmonsiaPasteurina , FT4A as Aspergillus niger , FT5B as Penicillium sp. , BT1 as Monococcusechinophorus , BT2 as Bacillus subtillus , BT3 as Streptococcus sanguis and BT4 as Pseudomonas flourescens. Based on their morphological and cultural characteristics F26 and F44 were tentatively identified as Trichodermaviride and Aspergillus niger , respectively (Kausar et al., 2010 ). CONCLUSION The indigenous fungal and bacterial species were isolated and purified from soil of different cropping management scenarios. As total of 15 isolates were selected according to their morphology. Then, after in-vivo and in-vitro screening, it was culminated that the potential lignocellulolytic strains were identified FT1B as Aspergillus flavus , FT2A as EmmonsiaPasteurina , FT4A as Aspergillus niger , FT5B as Penicillium sp. , BT1 as Monococcusechinophorus , BT2 as Bacillus subtillus , BT3 as Streptococcus sanguis and BT4 as Pseudomonas flourescens from both primary and secondary evaluation. The rapid degradation of lignocellulosic biomass by autochthonous microbes can make it possible to avoid in-situ burning of crop residues and use of a typical zero-till machine for timely sowing of wheat into combined harvested rice fields. The study’s outcomes are expected to have far-reaching in-situ implications and paving the way for future research. Declarations Acknowledgements: We are thankful to laboratory technical staff member of Department of Agronomy, Faculty of Agricultural Sciences, University of the Punjab, Lahore for providing facilities during the study time. Competing Interest: The authors declare that there is no conflict of interest Author Contributions: This manuscript was written through the contributions of all authors. Ammara Fatima: design the research plan, help in chemical analysis and write up Adnan Zahid: design the research plan, help in chemical analysis and write up, Sajid Ali: support in chemical analysis and write up. Waheed Anwar: chemical analysis, arranging the data and write up. Amina Arshad: Support in collecting the sample and chemical analysis, Rutaba Zia: support in collecting the sample and chemical analysis and write up Sana Imdad: Support in collecting the sample and chemical analysis, Muddasir A Dar: arranging data and write up. Availability of data and material : The writers will provide the information you need on the data that backs up the study's conclusions. Funding : The present study did not receive any funding. So, it is not applicable for the current work. Conflict of interest : The authors declare that they have no conflict of interest. Ethical approval not applicable in the present work. Consent to participate All individuals taking part in the study gave their informed permission. 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17:28:10","extension":"xml","order_by":66,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167619,"visible":true,"origin":"","legend":"","description":"","filename":"862306dafcaa4011849bad4304d749581structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/c032c8b0504199c278be23e0.xml"},{"id":92019099,"identity":"d69d6885-8c8a-4bf3-8acb-53fea133977c","added_by":"auto","created_at":"2025-09-23 17:12:10","extension":"html","order_by":67,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188198,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/949f1fa8a3295820272c9aed.html"},{"id":92019292,"identity":"ec8fb132-c38c-4e8b-a792-cb71e9d38512","added_by":"auto","created_at":"2025-09-23 17:20:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120832,"visible":true,"origin":"","legend":"\u003cp\u003eThe macroscopic characters of purified fungal colonies and y showed the defined bacterial colony\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/04cf0c7bd93ebb0ee956a6fc.jpg"},{"id":92019026,"identity":"43d77cfd-757f-49a6-b7e6-7640d9c2f464","added_by":"auto","created_at":"2025-09-23 17:12:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46224,"visible":true,"origin":"","legend":"\u003cp\u003eFungal isolates and their closest reference sequences obtained from NCBI are shown in a phylogenetic tree based on ITS region sequences. The isolates with the codes FT4A, FT2A, FT1B, and FT5B cluster with the respective reference strains, indicating both intra- and interspecific genetic diversity among the fungi examined and validating their taxonomic position.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/100bfd56ea6ace4e708bdf9f.jpg"},{"id":92019027,"identity":"f1c49c84-3e2c-4828-9c69-bced0c14bb32","added_by":"auto","created_at":"2025-09-23 17:12:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49282,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on 16S rRNA gene sequences showing the evolutionary relationship among fungal isolates and their closest reference sequences retrieved from NCBI. Bacterial Isolates labeled with specific codes monococus echinophous BT1, \u003cem\u003eBacillus subtilis\u003c/em\u003e BT2, \u003cem\u003eBacillus subtilis\u003c/em\u003e BT3, and \u003cem\u003ePseudomonas fluorescens \u003c/em\u003eBT4 cluster with corresponding reference strains, confirming their taxonomic placement and illustrating both intra- and interspecific genetic diversity among the sampled fungi.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/38aea037384d8149746e7ffd.jpg"},{"id":92019029,"identity":"01b47cfd-506d-4b1b-82c1-eca22eb0dc83","added_by":"auto","created_at":"2025-09-23 17:12:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29130,"visible":true,"origin":"","legend":"\u003cp\u003eIn plate media (1) a, b, c and d showed the max hydrolysis of cellulose by Fungal Cellulolytic Enzymes. (2) showed the hydrolysis of cellulose by Bacterial Cellulolytic Enzymes. (3 )a and b showed the max Polyphenol oxidation by fungal Lignolytic Enzyme.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/ab1528d2a1ce8909b4187933.jpg"},{"id":92019293,"identity":"18fcaa84-0cc5-4fc8-aa1e-eac7a77b5299","added_by":"auto","created_at":"2025-09-23 17:20:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110823,"visible":true,"origin":"","legend":"\u003cp\u003egraphs (a \u0026amp; b) showed the significant values of potential lignocellulolytic strains in CMCase Enzymatic Activity (Fungal strain (T4A) and Bacterial strain (T4) have highest hydrolysis strength), graphs (c \u0026amp; d) showed that the significant results of FPase Enzymatic Activity of both Fungal and Bacterial Strains and Graph e showed the T4A of fungal strain has max lignolytic activity.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/fc91e7ba459dce34b89d59b4.jpg"},{"id":92020012,"identity":"58775369-8475-410c-8306-66372d6b77db","added_by":"auto","created_at":"2025-09-23 17:28:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59505,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopically magnified (40X) pictures (a), (b) \u0026amp; (c) of fungal colonies and (d) \u0026amp; (e) of bacterial colonies\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/e23082d0fe8c1f31b175e693.jpg"},{"id":93769408,"identity":"7a320da3-96c2-4b51-b2af-cc5b1786f94d","added_by":"auto","created_at":"2025-10-17 11:32:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1783003,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7533898/v1/212e8a82-2c3c-49a4-9645-9e24819384be.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening and Characterization of Lignocellulolytic Microbes from the Tillage Ecosystems for Sustainable Valorization of Agro-waste","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRecent challenges to food security, driven by climate change, limited arable land, and soil degradation, are exacerbated by intensive agricultural practices and the widespread use of agrochemicals, which have boosted global production (Zarezadeh et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, these practices have led to the accumulation of significant organic waste, causing environmental pollution, soil health deterioration, and increased public health risks. Traditional waste management methods, including burning, landfilling, and chemical degradation, are inefficient, energy-intensive, and harmful to the environment (Popp et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The nexus between waste accumulation and deteriorating soil health underscore the urgency in addressing the rising threat to global food security (Jiang \u0026amp; Zhou, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), aligning with the United Nations' Sustainable Development Goals (Nations, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). With the global population projected to reach 10\u0026nbsp;billion by 2050, the need for sustainable agricultural systems is urgent, particularly in arid regions that support a third of the world\u0026rsquo;s population. As a result, there has been a shift towards sustainable, eco-friendly agri-technologies that offer value-added solutions for organic waste management, aiming to address pollution and support long-term environmental health (Dar et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCrop residues(CRs) which remain after harvest, are significant renewable resources and by-products of agriculture production. Being rich in macro- and micronutrients, CRs are pivotal for plant growth. Further, CRs account for approximately 40% of the total dry biomass, they are crucial for maintaining the stability of agricultural ecosystems (Sharma et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Amendment of CRs enhances organic matter content, boosts crop productivity, improves soil nutrients, and reduces essential nutrient depletion. These CRs are majorly composed of lignocellulose produced in tremendous quantities worldwide (Singh et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent estimates suggest that global lignocellulose biomass production reaches approximately 181.5\u0026nbsp;billion tons annually, yet only 8.2\u0026nbsp;billion tons are currently utilized across various applications (Mujtaba et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe lignocellulosic biomassis composed of proteins, salt, minerals, ash, lignin (an aromatic polymer), pectin (a heteropolysaccharide), carbohydrates (such as cellulose and hemicellulose), and other compounds, the compositions and proportions of lignocellulose compounds vary among different plants (Dar et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dar et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Plant cell wall layers contain lignin, cellulose, and hemicellulose as the middle lamella of a plant's cell wall contains the highest concentration of lignin, and the S2-layer of the secondary wall is particularly rich in cellulose (Manavalan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several organisms are known to degrade and utilize lignocellulosic biomass known as natural biomass utilization systems (Dar et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In nature, the degradation of lignocellulosic biomass occurs through the coordinated action of various microorganisms, predominantly fungi and bacteria, which secrete a range of cellulolytic and hemicellulolytic enzymes under both aerobic and anaerobic conditions (Ali et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Dar et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among these bio-resources bacteria and fungi are preferred biocatalysts due to their ease of cultivation, short growth periods and easy to bioengineer for enhanced activities (Xie et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). During the last decade, cellulase-producing bacteria have been isolated from diverse environments, including composting plants, decaying plant material from forestry or agricultural waste, ruminant feces, soils, organic matter, and the gastrointestinal tracts of animals (Sun et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although numerous cellulolytic bacteria have been documented across diverse ecological niches, the efficient and economically viable bioconversion of lignocellulosic biomass into commodity products remains a significant challenge (Adegboye et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, there is an urgent need for bio-prospection aimed at identifying more effective bacterial strains to enhance the bioconversion efficiency into value-added products. Isolating and purifying native bacteria and fungus from agricultural soils and decaying organic matter was the goal of this investigation. It used both primary and secondary screening techniques to assess their enzymatic activity and biodegradation potential. Finding viable microbial strains that could improve in-situ agricultural residue breakdown for quick, economical, and ecologically friendly composting methods was the aim.\u003c/p\u003e\u003cp\u003eTo reduce the negative effects of in situ burning and to maximize the use of various crop residues, an affordable and environmentally friendly residue management approach should be adopted (Shukla et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). An eco-friendly management strategy that uses lignocellulose-degrading microbes to hasten the degradation of CR is an alternative to avoiding environmental pollution (Garg, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). An effective method for accelerating the potential of lignocellulose degradation in agricultural residue is the inoculation of ligno-cellulolytic microorganisms. Numerous microbes including bacteria and fungi can produce enzymes for the breakdown of lignin, cellulose and hemicelluloses (Manavalan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt has become necessary to isolate and inoculate rice straw-degrading microbes to accelerate the decomposition of rice straw prior to wheat seeding. This will enable the use of standard zero-till planters and minimize the negative impacts on wheat growth. Even though residue-degrading bacteria and fungi are already present in soil, the amount of these fungi varies substantially between fields and locations based on environmental, edaphic, and management factors (Choudhary et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). According to our hypothesis, field inoculation will increase the population density of fast-degrading autochthonous fungi once they have been isolated and grown in fields under Rice-Wheat (RW) systems.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSoil Sample Collection:\u003c/h2\u003e\u003cp\u003eSoil samples were collected from long term residue management and conservation tillage experiment at Rice Research Institute (31\u0026deg;72\u0026rsquo; N, 74\u0026deg;28\u0026rsquo; E, Slope 1.8%), at Kala Shah Kaku (RRI, KSK), Shiekhupura, and Punjab, Pakistan. The five different tillage scenarios were based on cropping management, combined tillage and crop leftover management practices. In scenario-I; conventional tillage practices were used, scenario-II was led to no burning and ploughing, scenario-III was zero-tillage and non-puddled rice, scenario-IV was zero-tillage with puddled rice and scenario-V had conservation tillage (Zahid et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Soil samples were packed in the sterile polythene bags, labeled as T1, T2, T3, T4 and T5, respectively as per the scenario. After sample collections, the soil samples were transferred to the lab and maintained at 4 ℃.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation and Purification of Microbes:\u003c/h3\u003e\n\u003cp\u003eMicrobes were isolated from these soil samples to screen their lignocellulolytic potential. Serial dilution technique was used to isolate the fungal and bacterial strains from each five-soil samplevizT1, T2, T3, T4 and T5. After 10⁻⁵ dilutions, 1mL of last diluted soil sample was poured drop wise on already prepared PDA media plates for fungi and LBA media plates for Bacteria. PDA media plates were incubated at 28˚C for maximum 7\u0026ndash;10 days and LBA media plates were incubated at 37˚C for 48 h. The microbial growth was observed within these days by following previous methods (NAYAK et al., 2017; Shinde et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the basis of morphology, 2 different fungal colonies and 1 bacterial colony was isolated from each soil sample plate and transferred antiseptically on already prepared pertinent media plates to obtain pure culture. All plates were accurately labeled with different code name according to soil sample to avoid any uncertainty; FT1A, FT1B, FT2A, FT2B, FT3A, FT3B, FT4A, FT4B, FT5A and FT5B for fungal colonies and BT1, BT2, BT3, BT4 and BT5 for bacterial colonies. Purified colonies without any contamination were preserved in refrigerator at 4˚C for screening and identification (NAYAK et al., 2017; Shinde et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMolecular Identification of Microbes:\u003c/h3\u003e\n\u003cp\u003eFor bacterial and fungal Identification DNA extraction was conducted using the cetyltrimethylammonium bromide (CTAB) method (Schenk et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For fungal species, ITS primer were used. PCR amplifications for ITS, initial denaturation for 5 min at 95\u0026deg;C, followed by 35 cycles of denaturation at 94\u0026deg;C (30 s), annealing temperature at 56\u0026deg;C or 59\u0026deg;C (30 s), and extension at 72\u0026deg;C for 1 min. The amplification was accomplished with one additional step of final extension at 72\u0026deg;C for 10 min.(Asis et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The PCR conditions for Bacteria were 3 min at 95\u0026deg;C for 32 cycles, 1 min at 94\u0026deg;C, 1 min at 56\u0026deg;C, 2 min for 72\u0026deg;C, and 10 min for 72\u0026deg;C with 4\u0026deg;C intervals. After sequencing, the obtained sequences were blasted using the online tool NCBI Blast (Chukwuma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phylogenetic tree was made of different fungal and bacterial isolates by using MEGA X software.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis of Microbes.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe bacterial isolate 16S regions and the fungal isolates' ITS (Internal Transcribed Spacer) regions, as well as closely related reference sequences that were obtained from the NCBI GenBank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ncbi.nih.gov\u003c/span\u003e\u003cspan address=\"http://ncbi.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), were used to create a phylogenetic tree (Asis et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To guarantee the clustering pattern's dependability, the Neighbor-Joining technique was used in MEGA X for the analysis, which included 1000 bootstrap replications. MEGA X software was used to construct phylogenetic tree.\u003c/p\u003e\n\u003ch3\u003eIn-vitro Primary Screening of Potential Lignocellulolytic Microbes:\u003c/h3\u003e\n\u003cp\u003ePrimary Screening also known as qualitative screening was employed to determine the potential of the isolated microbes. To accomplish this, qualitative agar plate method was used for primary screening of lignocellulolytic fungi and bacteria. The appearance of the hydrolysis zone formed after the growth of the microorganisms was deemed as positive for lignocellulose degradation (Gahfif et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCellulolytic Degradation:\u003c/h3\u003e\n\u003cp\u003eThe cellulolytic or endoglucanase activity was tested by using the CMC agar media. The media was prepared with the following constituent\u0026rsquo;s g/500mL; 0.5g yeast extract, 13g carboxymethyl cellulose, 1.5g agar and trace elements 0.5g MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 0.1g KCL and 0.5g NH\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (Hankin \u0026amp; Anagnostakis, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) with modifications. Purified colonies of both microbes were inoculated on the CMC agar media plates (Choudhary et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kausar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThen, after their incubation and proper growth 1% Congo red dye solution was flooded in the media plates for 20 min. Subsequently, the plates were drained and washed with 1M NaCl solution for 20 min. A CMC clearance zone was appeared around fungal and bacterial colonies indicating cellulolytic potential. This clear zone was measured by the following formula to calculate the Enzymatic Index (EI) (Marđetko et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Namnuch et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Enzymatic\\:Index\\:\\left(EI\\right)=Diameter\\:of\\:hydrolysis\\:zone/Diameter\\:of\\:colony$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eLignolytic Degradation:\u003c/h2\u003e\u003cp\u003eOxidative test was used to check the lignin degradation of fungi. Phenol oxidase was considered as a key element of lignin degradation. Tannic Acid agar media was used to screen the lignin degradation capacity of fungi by verifying the presence of polyphenol oxidase. Tannic Acid agar media was prepared by adding 7.5g malt extract agar, 10g agar and 2.5g Tannic acid under the sterile conditions and protective method (Dabhi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Thormann et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Then, purified fungal colonies were inoculated on these Tannic Acid agar media plates and incubated at 28˚C in dark place for 7-10days. The brownish or greenish color zone formation around the fungal colonies was the result of polyphenol oxidase degradation (Choudhary et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kausar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn-vitro Secondary Screening of Potential Lignocellulolytic Microbes:\u003c/h3\u003e\n\u003cp\u003eSecondary screening is the quantitative estimation of lignocellulolytic enzymes of fungal and bacterial strains. Basal broth media was used for the production of these enzymes. Under submerged fermentation, broth media was prepared by using the following constituents; CMC, wheat residue, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, Urea, MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, (NH\u003csub\u003e4\u003c/sub\u003e)2SO\u003csub\u003e4\u003c/sub\u003e, Peptone, yeast Extract, Tween-80, FeSO.7H\u003csub\u003e2\u003c/sub\u003eO, MnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO and ZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO. All these ingredients with concentration g/1100 given in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were added in to distilled water (Awadalla et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cyrus \u0026amp; Juwon, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eConcentrations of the components involved in the preparation of Broth Media.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIngredients\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentration (g/1100)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarboxymethyl Cellulose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ewheat residue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.2g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUrea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(NH\u003csub\u003e4\u003c/sub\u003e)2SO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.54g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeptone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.275g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eyeast Extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.11g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTween-80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.1mL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0055g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00176g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00154g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAfter the inoculation of both microbes, flasks with fungal inoculation were placed in to shaking incubator at 28˚C and 160 rpm for 7 days and with bacterial inoculation were placed in to shaking incubator at 37˚C and 160 rpm for 24 h. Then, the incubated broth was filtered and centrifuged at 10,000 rpm for 10 min at 4˚C. The clear supernatant was accumulated and stored at 4˚C. These crude enzymes were further used for the quantitative lignocellulolytic analysis of these enzymes by CMCase, FPase and Lacase assay techniques (Choudhary et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Namnuch et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCMCase and FPase Assay:\u003c/h3\u003e\n\u003cp\u003eCMCase and Filter Paperase assay technique were used for Endo-β-1,4-Glucanase test. The reported procedure of (Ghose, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Miller, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1959\u003c/span\u003e) was followed by using 2% CMC and filter paper strips of size 1cm x 6cm as a substrate respectively. Sodium citrate buffer and substrate was thoroughly mixed in test tubes and placed in to incubator at 50˚C for 30 minutes. DNS (3 5- dinitrosalicylic Acid) (Miller, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1959\u003c/span\u003e; Sivaramanan, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) added to consummate the enzymatic reaction and for measuring the reducing sugar.\u003c/p\u003e\u003cp\u003eUV spectrophotometer was used to measure the optical absorbance of reducing sugar of each sample at 540nm against spectro zero. The readings of optical absorbance of main samples were subtracted from their enzyme blanks and spectro zero. These readings were compared and plotted with glucose standard curve. Under the assay conditions, the amount of enzyme that was required to release 1 \u0026micro;mole of reducing sugar per min was one unit of enzyme activity (Awadalla et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cyrus \u0026amp; Juwon, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Cellulase Activity was calculated as per formula; Cellulase activity= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\mu\\:}\\text{m}\\text{o}\\text{l}\\text{e}\\text{s}\\times\\:{\\text{V}}_{\\text{t}}/{\\text{V}}_{\\text{s}}\\times\\:\\text{t}\\)\u003c/span\u003e\u003c/span\u003e (Adney \u0026amp; Baker, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLaccase Assay:\u003c/h2\u003e\u003cp\u003eThis assay technique was based on the oxidation of 2,2\u0026prime;-azino-bis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) method used to quantify the lignolytic potential of fungal enzymes. The reaction mixture was prepared by adding 10mM ABTS and the absorbance of the mixture was measured by using UV-spectrophotometer at 420nm. (NAYAK et al., 2017; Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) The amount of enzyme that was needed to oxidize 1 \u0026micro;mole of ABTS per min with molar absorbance 360000 M ⁻\u003csup\u003e1\u003c/sup\u003e cm⁻\u003csup\u003e1\u003c/sup\u003e is the one unit of enzyme activity (Marđetko et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Laccase enzyme was measured by the following formula (Baltierra-Trejo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e):\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{E}\\varvec{n}\\varvec{z}\\varvec{y}\\varvec{m}\\varvec{e}\\:\\varvec{A}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{v}\\varvec{i}\\varvec{t}\\varvec{y}\\frac{1\\varvec{U}}{\\varvec{m}\\varvec{l}}\\:=\\frac{\\varDelta\\:\\varvec{A}\\varvec{b}\\varvec{s}\\times\\:\\varvec{V}\\varvec{t}}{\\in\\:\\:\\times\\:\\varvec{I}\\varvec{n}\\varvec{c}\\varvec{u}\\varvec{b}\\varvec{a}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\varvec{t}\\varvec{i}\\varvec{m}\\varvec{e}\\:\\times\\:\\varvec{V}\\varvec{e}\\:\\times\\:\\varvec{p}\\varvec{a}\\varvec{t}\\varvec{h}\\:\\varvec{l}\\varvec{e}\\varvec{n}\\varvec{g}\\varvec{t}\\varvec{h}}.$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of Lignocellulolytic Microbes:\u003c/h2\u003e\u003cp\u003eFresh fungal and bacterial colonies were firstly identified macroscopically by their traits of size, shape, color and growth. Then, slides of all cultures were prepared to analysed them microscopically under Microscope Nikon Eclips E200 to see hyphal, spores and conidial shapes and different spherical, spiral and other bacterial shapes by 10X and 40X lenses and identified by various keys (John I Pitt \u0026amp; Ailsa Diane Hocking, 2009).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003eRandomized Control design was used for all experiments with three replications. The recorded data was statistically analyzed by ANOVA using (Statistics 8.1) and Least Significant Difference (LSD) was applied to find the significance between the different microbial colonies.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and Purification of Fungi:\u003c/h2\u003e\u003cp\u003eAs total of 10 fungal isolates and 5 bacterial isolates were isolated on the basis of different morphology, 3 isolates FT1A, FT1B and BT1 from the Scenario-I soil sample that was labeled as T1, 3 isolates FT2A, FT2B and BT2 from the Scenario-II (T2), 3 isolates FT3A, FT3B and BT3 from Scenario-III (T3), 3 isolates from Scenario-IV (T4) and 3 Isolates from Scenario-V soil sample (T5). These isolates were selected on their different dominant characteristics of color and shape given in the Table. 2 and manifested in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDominant characters of microbial colonies for their selection and purification.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eColor of Colonies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eShape of Colonies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTexture of Colonies\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT1A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite to Greyish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 Round\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCottony\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT1B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGreen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRounds\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSpore or granular\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT2A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePure white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRound\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eThick Hairy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT2B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight Green \u0026amp; white pink corners\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWrinkle Round\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHairy \u0026amp; spore\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT3A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight Pink white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIrregular\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWeak Hairy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT3B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDark Greenish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWrinkle Round\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evelvet\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT4A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePure Black\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRound\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSpore or granular\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT4B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGreen \u0026amp; white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRound\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHairy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT5A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight Green, white \u0026amp; pink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWrinkle Round\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHairy and spore\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT5B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGreyish\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWrinkle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003evelvety\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCreamy White\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eComma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSoft Creamy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGreyish White\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBacilli\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSoft Creamy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight Brown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCocci\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSoft Creamy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYellow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCylindrical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSoft Creamy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eClub Rod\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSoft Creamy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Tree of Fungal Isolates Based on ITS Sequences\u003c/h2\u003e\u003cp\u003eThe phylogenetic tree was constructed based on the alignment of ITS (internal transcribed region) gene sequences to determine the evolutionary relationships among the isolated fungal strains. \u003cem\u003eAspergillus niger, Aspergillus flavus, Penicillium sp., and Emmonsia sp.\u003c/em\u003e were among the closely related reference strains that the isolates clustered with, according to the analysis. Accurate identification and evolutionary relation were confirmed by this tree, which shows genetic similarity between the isolates and their corresponding reference sequences.\u003c/p\u003e\u003cp\u003eThe resulted fungal isolates tree clearly showed that the isolates group within distinct clades corresponding to their respective genera and species, demonstrating high similarity with known reference strains such as \u003cem\u003eAspergillus niger\u003c/em\u003e, and \u003cem\u003ePenicillium spp.\u003c/em\u003e The close clustering of fungal isolates with authenticated reference sequences confirms their molecular identity and evolutionary placement. This analysis not only validates morphological identification but also provides insights into the genetic diversity and relatedness of the studied fungal community. The presence of \u003cem\u003eEmmonsia sp.\u003c/em\u003e as a separate branch reflects their genetic divergence from \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003ePenicillium.\u003c/em\u003e Overall, the phylogenetic analysis effectively differentiates between species and underscores both inter- and intra-species diversity among the sampled fungi, supporting the taxonomic classification and genetic variations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Tree of Bacterial Isolates Based on 16S rRNA Sequences\u003c/h2\u003e\u003cp\u003eThe phylogenetic tree was constructed based on the alignment of 16S rRNA gene sequences to determine the evolutionary relationships among the isolated bacterial strains. The analysis revealed clustering of the isolates with closely related reference strains, including \u003cem\u003eStreptococcus sanguis\u003c/em\u003e, \u003cem\u003ePseudomonas fluorescence\u003c/em\u003e, and \u003cem\u003eBacillus subtilis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe 16S rRNA gene sequence analysis confirmed the molecular identity of the bacterial isolates obtained in this study. The phylogenetic tree showed that \u003cem\u003eBacillus subtilis\u003c/em\u003e BT2 clustered closely with reference strains of \u003cem\u003eBacillus subtilis\u003c/em\u003e, indicating a high degree of similarity and confirming its identification. Similarly, \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e BT4 grouped with authentic strains of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e, demonstrating strong evolutionary relatedness. Interestingly, \u003cem\u003eBacillus subtilis\u003c/em\u003e BT2 showed close alignment with \u003cem\u003eStreptococcus sanguis\u003c/em\u003e BT3, suggesting genetic similarity or possible horizontal gene transfer events. Overall, the clustering pattern verified that the isolates belonged to well-established bacterial taxa and highlighted their evolutionary relationships with closely related species deposited in the NCBI database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003ePrimary Screening of Lignocellulolytic Microbes:\u003c/h2\u003e\u003cp\u003eThe Purified isolates were then screened on solid plate media to check their cellulose and lignin degradation capability. The zones were appeared around colonies that showed their ligocellulolytic potential. These zones were measured by a cm scale to compare the colonies potential.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eCellulolytic Degradation:\u003c/h2\u003e\u003cp\u003eThe clear zone or yellow opaque color zone was formed around the purified colonies on CMC agar media by applying congo red dye solution. This clear zone was visualized the hydrolysis of cellulose by both fungal and bacterial enzymes. All the colonies were showed the zone formation with the varying capability of cellulose breakdown. They were showed their cellulolytic activity in the range 0.1cm to 1.6cm. It was estimated by measuring the one-side length (Radius) of the zone formation. Fungal isolates FT4A, FT1A, FT1B and FT3A were showed the highest length of cellulolytic zone formation that was 1.5 cm, 1.1 cm, 1.2 cm and 1 cm respectively. Bacterial isolates BT2 and BT4 showed the highest length of cellulolytic zone formation that was 1.5 cm and 1 cm respectively. The length of other isolates was mentioned in the Table.3.2 and visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eLignolytic Degradation:\u003c/h2\u003e\u003cp\u003eThe greenish or brownish color zone was formed around the purified fungal colonies on Tannic Acid agar media. This zone formation was the hydrolysis of polyphenol oxidase by the fungal enzymes that demonstrated the lignin activity. All the fungal colonies were not showed the zone formation; only few isolates had varying ability of lignin breakdown. They were showed their lignolytic activity in the range 0.1cm to 1cm. It was estimated by measuring the one-side length (Radius) of the zone formation. Fungal isolates FT2A, FT4A and FT5B were showed the highest length of lignolytic zone formation and their values were 1cm, 0.9cm and 0.8cm respectively. The length of zone formation of other isolates was mentioned in the Table.3 also showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eTable.3. Qualitative Screening of Potential Lignocellulolytic Microbes on Agar Media Plates.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSr. No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMicrobial Strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSource of the strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLignocellulolytic Activity Check\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eEnzymatic Index of Lignocellulolytic Zone Formation (cm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCellulose Degradation on CMC agar (Radius)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLignin Degradation on Tannic Acid agar\u003c/p\u003e\u003cp\u003e(Radius).\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT1A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT1B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT2A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+,+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT2B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT3A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT3B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT4A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT4B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT5A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFT5B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil from S5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+, -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil S2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil S3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil S4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSoil S5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eSecondary Screening of Lignocellulolytic Microbes:\u003c/h2\u003e\u003cp\u003eQuantitative Screening of all fungal enzymes was measured on CMC liquid broth media amended with wheat straw. CMCase, FPase and Lacase assays were performed for the quantification of enzyme activity of Lignocellulolytic fungi.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eCMCase Assay:\u003c/h2\u003e\u003cp\u003eThe 1U/mL/min cellulolytic enzyme activity of all microbial enzymes was measured by using slope value and enzyme activity calculations. All the enzymes had cellulolytic activities with different concentrations. Cellulolytic activity of CMCase assay was ranged from 1.1 U/mL to 11.6 U/mL as given in Table. 4. Fungal strains FT1A, FT1B and FT4A have the highest cellulolytic values 5.8 U/mL, 11.6 U/mL and 6.7 U/mL in CMCase assay. Bacterial strains BT4 and BT2 have the highest cellulolytic values 6.75 U/mL and 2.19 U/mL in CMCase assay. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Graphs (a \u0026amp; b) represented the significant difference of both bacterial and fungal hydrolytic enzyme activities.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eFPase Assay:\u003c/h2\u003e\u003cp\u003eThe 1U/mL/min cellulolytic enzyme activity of all enzymes in FPase was also measured by using slope value and enzyme activity calculation. In FPase all the enzymes also had cellulolytic activities with different concentrations. Cellulolytic activity of FPase assay was ranged from 0.04 U/mL to 7.7 U/mL as given in Table.4. Fungal strains FT2B and FT1B have the highest cellulolytic values 7.7U/mL and 6.9 U/mL in FPase assay. Bacterial strains BT5 0.62U/mL and BT2 0.14U/mL have the highest cellulolytic values. FPase assay values were different from CMCase assay. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. and Graph (c \u0026amp; d) showed the significant values of FPase assay.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLaccase Assay:\u003c/h2\u003e\u003cp\u003eLaccase assay was used to measure the lignin activity of all fungal enzymes. Enzyme activity 1U/mL/ min was calculated by the oxidation of substrate ABTS. Fungal enzymes showed very less lacase activities. The range of activity was from 0.089 U/mL to 1.14U/mL. The highest value of lacase was showed by FT4A and FT5A had lowest value about negligible. Table. 4 and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e graphs displayed the significant ranges of quantitative lignolytic activity of fungi.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTable.4 Quantitative Screening of Potential Lignocellulolytic Microbes by submerged fermentation.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr.No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMicrobial Strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCMCase Activity\u003c/p\u003e\u003cp\u003e(1U/ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFPase Activity\u003c/p\u003e\u003cp\u003e(1U/ml/min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLaccase Activity\u003c/p\u003e\u003cp\u003e(1U/ml/min)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT1A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT1B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT2A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT2B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.012\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT3A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.095\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT3B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.954\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.087\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT4A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT4B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT5A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.089\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT5B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e14.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eIdentification of the isolated Lignocellulolytic Microorganisms:\u003c/h2\u003e\u003cp\u003eThe macroscopic characteristics were observed by their color, size, shape, texture and growth. The microscopic traits of fungi were observed by the shape of Hyphae (septate and non-septate) and shape of Conidia (regular, irregular, round and rough e.t.c) and bacteria was observed by their shapes: spherical (cocci), rod (bacilli), spiral (spirilla), comma (vibrios) or corkscrew (spirochaetes). These differentiating attributes were specified in Table. 5 and be revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMicroscopic characteristics of Lignocellulolytic Microbes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr.No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMicrobial Strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNature of Hyphae\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eConidia Shape\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eForm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMargin\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT1A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFilamentous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT1B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRough\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT2A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRound\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT2B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSpherical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT3A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlobose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT3B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIrregular\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT4A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlobose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT4B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRough\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT5A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIrregular\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFT5B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSeptate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSpherical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIrregular small\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUndulate\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIrregular small\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFlat\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRegular small\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLobate\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRegular large\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSmooth\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eBT5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIrregular small\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLobate\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMacroscopic and microscopic identification of potential lignocellulolytic strains revealed; FT1B as \u003cem\u003eAspergillus flavus\u003c/em\u003e, FT2A as \u003cem\u003eEmmonsiaPasteurina\u003c/em\u003e, FT4A as \u003cem\u003eAspergillus niger\u003c/em\u003e, FT5B as \u003cem\u003ePenicillium sp.\u003c/em\u003e, BT1 \u003cem\u003eas Monococcusechinophorus\u003c/em\u003e, BT2 as \u003cem\u003eBacillus subtillus\u003c/em\u003e, BT3 as \u003cem\u003eStreptococcus sanguis and\u003c/em\u003e BT4 as \u003cem\u003ePseudomonas flourescens (John I Pitt \u0026amp; Ailsa D Hocking, 2009).\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eLignocellulosic waste is the most abundant biorenewable biomass on earth, and its hydrolysis releases highly valued reducing sugars. However, the presence of lignin in the biopolymeric structure makes it highly resistant to solubilization thereby hindering the hydrolysis of cellulose and hemicellulose. Microorganisms are known for their potential complex enzymes that play a dominant role in lignocellulose conversion (Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plant cell walls are degraded and assimilated by microorganisms in a complex process that involves the cooperative action of many different organisms and the enzymes outside of cells. In plants, the cell wall primarily complex polysaccharides (cellulose, Hemicellulose and pectin) which together make up a dense substance, coated phenolic polymer with multiple uses (M'barek et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The lignocellulosic biomass is broken down by cellulases to produce fermentable sugars. Natural lignocellulose must be saccharified enzymatically, and when working with lignocellulosic substrates, the use of cellulose-degrading enzymes is crucial (Donohoe \u0026amp; Resch, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBiomass burning after crop harvesting is the most opted choice for farmers in order to manage residues. Though it is an easier option but it is not eco-friendly and has negative effects on the environment such as release of greenhouse gases and fine particulate matter. An alluring alternate strategy was used of Lignocellulolytic microbes. So, present study is aimed to come up with a viable alternative to crop residue burning as a way to attain sustainable alternative to protect the environment and explore the ability of potent microbes to selectively delignify.\u003c/p\u003e\u003cp\u003eTotal 15 microbial isolates were isolated on the basis of different morphology from cropping management scenarios. The purified isolates were then screened on solid plate media to check their cellulose degradation capability. The clear zone or yellow opaque color zone was formed around the purified colonies on CMC agar media. All the colonies were showed the zone formation with the varying ability of cellulose breakdown. The fungal colonies showed their cellulolytic activity in the range 0.1cm to 1.6cm and bacterial colonies showed their activity in the range 0.5cm to 1.25cm.\u003c/p\u003e\u003cp\u003eSeveral researchers (Choudhary et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Keerthana et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported similar findings. Cellulase is an enzyme that the cellulolytic microorganisms make that can break down cellulose into monomeric or dimeric forms that are soluble in water and can be used as a carbon or food source for colony growth (Romsaiyud et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Five isolated microbial cultures (IMCs) demonstrated the development of clear zones around the inoculation site, which are easily visible after staining with Congo red dye. The seven active microbial cultures had zone formations with diameters ranging from 1.1 to 2.9 cm. The sample IMC 18's maximum zone was 2.9 cm, whereas the sample IMC 4's maximum zone was 2.1 cm. IMC 11 showed a moderate clear zone measuring 1.9 cm in diameter, with zone diameters of 1.2 cm for IMCs 6 and 17, and 1.1 cm for IMCs 1 and 20. The remaining 14 cultures had no discernible distinct zone (Shinde et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor lignolytic screening on agar media, fungal cultures were grown on Tannic acid agar media. All the fungal cultures were not showed the zone formation; only few isolates had varying ability of lignin breakdown. They were showed their lignolytic activity in the range 0.1cm to 1cm.\u003c/p\u003e\u003cp\u003eOn tannic acid media, isolate F44 developed a considerably (p 0.05) larger dark brown zone (84.71%). The tannic acid medium developed a dark brown zone, which demonstrated the fungal isolate's polyphenol oxidase (PPO) activity. In order to create dark brown complexes, which are essential for the breakdown of the phenolic molecule in lignin, polyphenol oxidase, a combination of monophenol oxidase and catechol oxidase, catalyzed the reaction between polyphenol and molecular oxygen. During the biodegradation of lignocellulosic materials, fungi have been demonstrated to create ligninolytic enzymes (Rodrigues et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). (Dinis et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) also reported on the activities of lignin peroxidases, manganese peroxidases, and laccase during solid state fermentation of lignocellulosic materials.\u003c/p\u003e\u003cp\u003eQuantitative screening of all microbial hydrolytic enzymes was measured on CMC liquid broth media amended with wheat straw. CMCase, FPase and Lacase assays were performed for the quantification of enzyme activity of Lignocellulolytic microbes. Fungal strains FT1A, FT1B and FT4A and Bacterial strains BT4 and BT2 have the highest cellulolytic values 5.8 U/ml, 11.6 U/ml, 6.7 U/ml, 6.75 U/ml and 2.19 U/ml respectively in CMCase assay. FT2B, FT1B, BT5 and BT2 have the highest cellulolytic values 7.7U/ml, 6.9 U/ml, 0.62U/ml and 0.14U/ml respectively in FPase assay. FT4A had highest lignolytic potential 0.14U/ml in Lacase assay and many isolates had least activity.\u003c/p\u003e\u003cp\u003eThe highest CMCase activity of IMC 18 was (0.26 IU/ml), followed by IMC 4 (0.21) and IMC 11 (0.16). IMC1 had the least amount of activity (0.05) and several cultures, such as IMC 6 (0.07), IMC 20 with 0.08 and 17 (0.09) were found to possess a moderate CMCase activity. When FPase utilization of the Whatman No. 1 filter to quantify activities IMC 18 once more shown the highest activity of IMC 4 was second highest at 0.14 IU/ml 0.11 MU/ml. IMC 6 displayed a low FPase activity (0.008), although IMC 1, 11, 17, and 20 did not placed in the middle. FPase activity was overall ranged between 0.02 and 0.14 for each of the seven colonies (Shinde et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThen, they were firstly identified macroscopically by their traits of size, shape, color and growth and microscopically by their traits of shape of hyphae and conidia shape. The potential Lignocellulolytic fungal colonies were identified FT1B as \u003cem\u003eAspergillus flavus\u003c/em\u003e, FT2A as \u003cem\u003eEmmonsiaPasteurina\u003c/em\u003e, FT4A as \u003cem\u003eAspergillus niger\u003c/em\u003e, FT5B as \u003cem\u003ePenicillium sp.\u003c/em\u003e, BT1 \u003cem\u003eas Monococcusechinophorus\u003c/em\u003e, BT2 as \u003cem\u003eBacillus subtillus\u003c/em\u003e, BT3 as \u003cem\u003eStreptococcus sanguis and\u003c/em\u003e BT4 as \u003cem\u003ePseudomonas flourescens.\u003c/em\u003e Based on their morphological and cultural characteristics F26 and F44 were tentatively identified as \u003cem\u003eTrichodermaviride\u003c/em\u003e and \u003cem\u003eAspergillus niger\u003c/em\u003e, respectively (Kausar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe indigenous fungal and bacterial species were isolated and purified from soil of different cropping management scenarios. As total of 15 isolates were selected according to their morphology. Then, after \u003cem\u003ein-vivo\u003c/em\u003e and \u003cem\u003ein-vitro\u003c/em\u003e screening, it was culminated that the potential lignocellulolytic strains were identified FT1B as \u003cem\u003eAspergillus flavus\u003c/em\u003e, FT2A as \u003cem\u003eEmmonsiaPasteurina\u003c/em\u003e, FT4A as \u003cem\u003eAspergillus niger\u003c/em\u003e, FT5B as \u003cem\u003ePenicillium sp.\u003c/em\u003e, BT1 \u003cem\u003eas Monococcusechinophorus\u003c/em\u003e, BT2 as \u003cem\u003eBacillus subtillus\u003c/em\u003e, BT3 as \u003cem\u003eStreptococcus sanguis and\u003c/em\u003e BT4 as \u003cem\u003ePseudomonas flourescens\u003c/em\u003efrom both primary and secondary evaluation. The rapid degradation of lignocellulosic biomass by autochthonous microbes can make it possible to avoid \u003cem\u003ein-situ\u003c/em\u003e burning of crop residues and use of a typical zero-till machine for timely sowing of wheat into combined harvested rice fields. The study\u0026rsquo;s outcomes are expected to have far-reaching \u003cem\u003ein-situ\u003c/em\u003e implications and paving the way for future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We are thankful to laboratory technical staff member of Department of Agronomy, Faculty of Agricultural Sciences, University of the Punjab, Lahore for providing facilities during the study time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u003c/strong\u003e\u0026nbsp; \u0026nbsp;The authors declare that there is no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eThis manuscript was written through the contributions of all authors. Ammara Fatima: design the research plan, help in chemical analysis and write up Adnan Zahid: design the research plan, help in chemical analysis and write up, Sajid Ali: support in chemical analysis and write up. Waheed Anwar: chemical analysis, arranging the data and write up. Amina Arshad: Support in collecting the sample and chemical analysis, Rutaba Zia: support in collecting the sample and chemical analysis and write up Sana Imdad: Support in collecting the sample and chemical analysis, Muddasir A Dar: arranging data and write up.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: The writers will provide the information you need on the data that backs up the study\u0026apos;s conclusions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: The present study did not receive any funding. So, it is not applicable for the current work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthical approval not applicable in the present work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent to participate All individuals taking part in the study gave their informed permission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdegboye MF, Ojuederie OB, Talia PM, Babalola OO (2021) Bioprospecting of microbial strains for biofuel production: metabolic engineering, applications, and challenges. 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Biomed Res Int 2021(1):5514745\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lignocellulose degradation, Cellulases, Lignin peroxidase, agro-waste, Bacteria, Fungi, Waste valorization","lastPublishedDoi":"10.21203/rs.3.rs-7533898/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7533898/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBurning of crop residues contributes significantly to air pollution, increases black carbon emissions, and accelerates climate change. Sustainable alternatives involve returning residues to the soil and applying lignocellulolytic microorganisms to speed up their breakdown, thereby supporting eco-friendly farming systems. This study focused on isolation and screening of the lignocellulolytic microbes particularly bacteria and fungi from tillage management scenarios as a viable alternative to crop residue burning. A total of 10 fungal and 5 bacterial strains were isolated in the form of pure colonies and their lignocellulolytic potential was screened by qualitative and quantitative screening. Primarily, the lignocellulolytic degradation was evaluated by using carboxymethylcellulose (CMC) and Tannic Acid (TA) agar media, on the basis of appearance of zone formation. Microbial strains FT4A, FT1B, BT4, BT2, FT2A and FT4A were showed the highest length of cellulolytic and lignolytic zone formation. Secondly, they were quantitatively screened by standard protocols of CMCase, FPase and Laccase enzyme assay techniques. FT1B and BT4 have the highest cellulolytic values 11.6 U/mL and 6.7 U/mL in CMCase assay. In FPase assay, FT2B and BT3 have the highest cellulolytic values 7.7U/mL and 0.14 U/mL. FT4A had highest lignolytic potential 0.14U/mL in Laccase assay and many isolates had least activity. The potential colonies which had significant results were identified as \u003cem\u003eAspergillus flavus, EmmonsiaPasteurina, Aspergillus niger\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003esp, \u003cem\u003eMonococcusechinophorus, Bacillus subtillus, Streptococcus sanguis\u003c/em\u003e and \u003cem\u003ePseudomonas flourescens\u003c/em\u003e. These species will be very useful if they apply in-situ crop residue degradation which ultimately improve the air quality, reduce environmental pollution and conservation of microbial biodiversity.\u003c/p\u003e","manuscriptTitle":"Screening and Characterization of Lignocellulolytic Microbes from the Tillage Ecosystems for Sustainable Valorization of Agro-waste","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 17:12:03","doi":"10.21203/rs.3.rs-7533898/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"430408be-bbd2-4ea7-ab2a-5d7a4bd9f65d","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-17T11:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 17:12:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7533898","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7533898","identity":"rs-7533898","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}