Evaluation of Cellulolytic Gut Bacteria Isolated from White Grubs (Holotrichia serrata and Leucopholis coneophora) and Their Utilization in Lignocellulose Degradation

Evaluation of Cellulolytic Gut Bacteria Isolated from White Grubs (Holotrichia serrata and Leucopholis coneophora) and Their Utilization in Lignocellulose Degradation | 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 Evaluation of Cellulolytic Gut Bacteria Isolated from White Grubs (Holotrichia serrata and Leucopholis coneophora) and Their Utilization in Lignocellulose Degradation Gatta Vis, KV Prakash, BN Ramesh, P Bhavani, KR Gagandeep This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4958316/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 Abstract The gut microbiota of insects plays a crucial role in digesting food, providing nutrients, and synthesizing enzymes. This approach is particularly relevant for degrading lignocellulosic biomass and managing waste. In Karnataka, the larvae of Holotrichia serrata and Leucopholis canephora are major crop pests, but the role of their bacterial communities in lignocellulose degradation has not been well studied. This study aimed to isolate and evaluate bacteria from these larvae for their ability to degrade lignocellulose.Approximately seventeen cellulolytic bacterial strains were isolated from the fermentation chamber of white grubs, primarily from the Firmicutes and γ-proteobacteria classes. Notable species included Bacillus, Enterobacter, and Klebsiella. Bacillus toyonensis strain LC3B1 demonstrated significant cellulolytic activity, with a cellulolytic index of 1.93 ± 0.037. The degradation of corncob powder was the highest (28.15 ± 1.56%), followed by that of paddy straw powder (31.45 ± 0.608%) and groundnut husk powder (33.25 ± 0.823%), indicating the strong ability of these powders to degrade agricultural residues. FTIR analysis of the substrate carboxymethyl cellulose (CMC) hydrolyzed by LC3B1 revealed decomposition products such as ketones, aldehydes, alcohols, and carboxylic acids. Scanning electron microscopy (SEM) revealed significant morphological changes and the formation of pores and tunnels in the treated biomass.The diverse cellulolytic capabilities of gut bacteria from white grubs, including those of the Bacillaceae, Enterobacteriaceae, and Pseudomonadaceae families, offer promising opportunities for lignocellulosic biomass degradation, biofuel production, and sustainable waste management. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Insects are among the most omnipresent and dominant organisms on Earth, constituting the largest and most diversified class within the phylum Arthropoda. This extensive diversity makes Insecta a significant and widespread group within the animal kingdom, characterized by their remarkable adaptability and ecological success. A critical aspect of insect biology is their symbiotic interactions with microorganisms, which range from mutualistic to parasitic relationships. Among these, the insect gut microbiota plays a pivotal role in aiding digestion and nutrient acquisition, forming complex and varied colonies of bacteria, fungi, and protozoa within the digestive system [ 1 , 2 ]. These symbiotic microorganisms are particularly important for the digestion of lignocellulosic biomass, a primary component of the plant cell wall, which many insects utilize as a food source. Lignocellulose, composed mainly of cellulose, hemicellulose, and lignin, is notoriously difficult to degrade due to its complex structure. Insects, however, have evolved highly specialized digestive systems, often complemented by their gut microbiota, to break down these complex carbohydrates. Notably, termites and certain beetles are known to harbor gut bacteria from the phyla Firmicutes and Proteobacteria, which facilitate cellulose degradation [ 4 , 5 ]. The gut microflora performs many functions, including synthesizing novel compounds and enzymes with therapeutic and industrial applications [ 6 – 8 ]. Moreover, microbiota is known to contribute to bioremediation and the degradation of plastics [ 9 , 10 ], as well as playing roles in disease prevention and pest management [ 11 , 12 ]. Despite extensive research on termites and beetles, there is a notable gap in understanding the cellulose-degrading capabilities of white grubs. White grubs, the larvae of scarab beetles, are polyphagous subterranean herbivores that pose significant agricultural challenges. The two white grub species, Holotrichia serrata and Leucopholis coneophora , which are major pests in Karnataka, India are the major focus of this study. These species are of particular interest due to their ability to feed on the roots of economically important crops, such as sugarcane and arecanut, which are rich in cellulose and nitrogenous compounds [ 16 – 19 ]. The present investigation aims to isolate and characterize the gut microbiota of these white grub species, with a specific focus on their potential to degrade lignocellulose. Understanding the symbiotic relationships between these insects and their gut microbiota not only contributes to the basic understanding of pest biology and host-microbiome co-evolution but also highlights the potential applications of these findings in bioengineering digestive enzymes for industrial use, including in biorefinery and biofuel production [ 20 – 25 ]. Material and methods Substrate Preparation Agricultural wastes such as paddy straw, groundnut husk, and corncob powder were collected from local fields at the University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bengaluru, Karnataka, India. The pretreatment of these agricultural wastes involved treating them separately with a mild alkaline solution (0.1 N NaOH, w/v). Following pretreatment, the samples were thoroughly washed with deionized water to achieve a neutral pH, air-dried, finely milled, and then sieved through a 2.0 mm screen to ensure uniform particle size. The pretreated substrates were either used immediately for hydrolysis experiments or stored in airtight containers for future use [ 27 ]. Insect sample collection The third-instar larvae of two white grub species, Leucopholis coneophora and Holotrichia serrata , were collected from infested fields in Moodabidri, Dakshina Kannada district, and Mahadeshwarapura, Mandya district of Karnataka, India, respectively. The identification of these white grub species was performed by Dr. K.V. Prakash, an entomologist, using binomial keys. The larvae were transported to the laboratory in sterilized, aerated plastic containers and were starved for 24 hours before dissection. Isolation of Gut Bacteria The larvae were rinsed in double distilled water for 30 seconds, followed by 70% ethanol for 60 seconds, and then rinsed again in double distilled water for 30 seconds to remove the disinfectant. The sterilized larvae were then dissected under laminar airflow using sterile microscissors to extract the gut. The fermentation chamber was isolated from the gut and carefully removed [ 28 ]. The dissected fermentation chamber was pooled into a 1.5 mL microtube containing 1.0 mL of phosphate-buffered saline (PBS, pH 7.4) and macerated using a sterile micropestle. The homogenized gut extracts were then enriched in Berg Minimal Salt media (BMS), which was supplemented with 2 g/L NaNO₃, 0.02 g/L MgSO₄·7H₂O, 0.02 g/L MnSO₄·H₂O, 0.5 g/L K₂HPO₄, 0.02 g/L FeSO₄·7H₂O, and 0.5 g/L CaCl₂·2H₂O, containing 1% (w/v) carboxymethyl cellulose (CMC) as the substrate [ 27 ]. The seeded medium was incubated at 37°C with shaking at 150 rpm for 48 hours. Following incubation, the culture broth was serially diluted in PBS (pH 7.4) to a final dilution of 10⁻⁷. One hundred microliters of each dilution were spread onto BMS-CMC agar plates and incubated at 37°C for 48 hours. Colonies were purified by repeated streaking on LB agar media, and the isolated strains were stored at -80°C in glycerol. Molecular Characterization and Identification of Isolated Bacterial Strains: Genomic DNA from the bacterial isolates was extracted following a modified protocol [ 29 ]. The bacterial isolates were cultured in LB broth and incubated overnight at 37°C with shaking. Approximately 1.5 mL of each culture was transferred to a microcentrifuge tube and centrifuged for 7 minutes. The supernatant was carefully removed, and the pellet was resuspended in TE buffer containing 100 mg/mL lysozyme, 20 mg/mL proteinase K, and 10% SDS. This mixture was incubated for one hour at 37°C. Following incubation, 5 M NaCl and CTAB solution were added, and the mixture was incubated at 65°C for 10 minutes. The sample was then washed with a mixture of chloroform and isoamyl alcohol (24:1), and the aqueous phase was transferred to a fresh tube. An equal volume of phenol:chloroform: isoamyl alcohol (25:24:1) was added, followed by centrifugation at 8,000 rpm for 5 minutes at 4°C. This step was repeated until a clear supernatant was obtained. DNA precipitation was achieved by adding an equal volume of chilled isopropanol, mixing gently, and incubating overnight at -20°C. The DNA was pelleted by centrifugation at 10,000 rpm for 20 minutes at 4°C, washed with 70% ethanol, air-dried, and finally dissolved in TE buffer. PCR amplification of the 16S rRNA gene was performed using the specific primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The thermal cycling conditions were as follows: an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 7 minutes. The PCR products were assessed for size and purity on 1.5% (w/v) agarose gels, after which they were purified and sequenced. The resulting partial sequences were aligned using the BioEdit tool [ 30 ]. BLAST analysis was conducted to identify closely related species in the NCBI database ( https://www.ncbi.nlm.nih.gov/ ). The sequences were further aligned using the ClustalW multiple sequence alignment tool [ 31 ], and a phylogenetic tree was constructed using the neighbor-joining method with the Kimura 2 evolutionary model [ 32 ]. The tree construction included a bootstrap value of 1000 replications and was performed using MEGA 11 software [ 33 ]. Screening of cellulose-degrading bacteria The bacterial isolates were inoculated onto BMS agar plates supplemented with carboxymethylcellulose (CMC) as the substrate and incubated at 37°C for 48 hours. To assess CMC degradation by the bacterial isolates, the plates were flooded with 0.1% Congo red solution and allowed to stain for 30 minutes. Excess Congo red was then removed, and the plates were treated with 1 M NaCl solution for 15 minutes. Degradation of the β-1,4 glycosidic bonds by cellulase activity prevented Congo red from binding to the cellulose polymer, resulting in clear zones on the medium [ 34 ]. Bacterial colonies that exhibited the most distinct and largest diameter zones of hydrolysis on the stained plates were selected for further investigations. The cellulolytic indices of the isolates were measured following a previously published protocol with slight modifications [ 35 ]. Cellulolytic index = (diameter of zone of clearance − diameter of bacterial colony)/ diameter of bacterial colony. Substrate Degradation Ratio Briefly, 1 mL of freshly grown bacterial culture (OD600: 0.5) was inoculated into BMS media supplemented with different substrates, followed by incubation in a rotary shaker at 37°C and 150 rpm for 7 days. After incubation, the mixture was centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected as a crude enzyme extract, which was stored at 4°C. To prepare the substrates, they were washed with 3 mL of acetic nitric reagent (a mixture of 150 mL 80% acetic acid, and 15 mL concentrated nitric acid). The substrates were then washed with distilled water for 5 minutes, followed by a wash with absolute ethanol for 10 minutes. The residues were subsequently dried at 60°C in a hot air oven [ 36 ]. The final weight of the substrates was measured , and the percentage of degradation was calculated using the following formula from Updegraff, 1969 [ 37 ]. Substrate Degradation (%) = {(Initial weight of substrate − Final weight of substrate) / Initial weight of substrate} ×100 Cellulase (β-1,4-Endoglucanase) Enzyme Assay The supernatant obtained from the previous experiment was used as the crude enzyme. For the enzyme assay, 0.5 mL of the supernatant was mixed with 1 mL of 0.05 M citrate buffer (pH 4.5) containing 1% CMC as the substrate. The reaction mixture was incubated in a water bath for 60 minutes. After incubation, 3 mL of DNS (dinitro salicylic acid) reagent was added to each tube containing the reaction mixture, which was then heated at 100°C for 5 minutes and subsequently cooled at 4°C for 5 minutes to stop the enzymatic reaction [ 38 ]. The absorbance of the reaction mixture was measured at 540 nm using a spectrophotometer, with a control sample as the baseline. The amount of reducing sugars produced was determined using a glucose standard. Enzymatic activity was expressed in units (U/mL), where one unit is defined as the amount of enzyme that releases 1 µmol of reducing sugars (measured as glucose) per mL per minute [ 39 ]. SEM Analysis of the Hydrolyzed Substrate The hydrolyzed and control substrates were characterized using Fourier-transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). A 1% bacterial inoculum was added to freshly prepared BMS liquid media containing 1000 mg of filter paper (FP) as the substrate, and the culture was incubated for 14 days at 150 rpm and 37°C. A control experiment was conducted under the same conditions but without bacterial inoculation. After the 14-day incubation period, the broth was centrifuged at 5000 rpm for 5 minutes to extract the biomass. For FESEM analysis, the samples were prepared following a previously published protocol with slight modifications [ 27 ]. The filter paper was washed with distilled water and dried overnight at 60°C. The samples were then sputter-coated with a 100 Å layer of gold in an argon gas atmosphere to enhance conductivity and reduce charging effects during scanning electron microscopy analysis. FTIR Analysis of the Hydrolyzed Substrate Fourier-transform infrared (FTIR) spectroscopy is a rapid and nondestructive technique for detecting functional groups in the mid-IR region. In this study, FTIR spectroscopy was used to analyze filter paper, which served as the sole carbon source for bacterial growth. After 14 days of bacterial treatment, filter paper samples, along with control samples, were mixed with 1000 mg of spectroscopy-grade potassium bromide (KBr) in an agate mortar and pressed into discs. The infrared (IR) spectra were recorded using an FTIR spectrometer in transmission mode, covering the range from 400 to 4000 cm⁻¹ [ 27 , 40 ]. SEM and FTIR analyses were conducted using the instruments available at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) in Bengaluru. Statistical Analysis The results for cellulolytic activity, substrate degradation, and enzyme assays were analyzed using descriptive statistics, analysis of variance (ANOVA), and Duncan's multiple range test (DMRT) to evaluate differences between factors at p = 0.05. The statistical analyses were performed using OP STAT [ 41 ], and the results were interpreted with Microsoft Excel (version 2021). FTIR data were plotted and analyzed using OriginPRO (version 2023b; OriginLab, Northampton, MA, USA) software. Results Isolation of Cellulolytic Gut Bacteria The morphological characteristics of bacterial isolates 1 to 7 from Leucopholis coneophora and 8 to 17 from Holotrichia serrata are provided in the Supplementary Data (Table S1). In total, seventeen bacterial isolates were obtained from the fermentation chambers (Figure 2) of two white grub species collected from distinct regions. Seven isolates were derived from L. coneophora , while ten were obtained from H. serrata (Fig. 1). Molecular characterization of cellulolytic gut bacteria DNA was extracted from all the samples, and 16S rRNA gene amplification using universal 16S rRNA primers produced high-quality amplicons, with sizes ranging from approximately 1450 bp (Figures S1 and S2). These PCR products were subsequently sequenced, and the resulting sequences were aligned using the BioEdit tool. The sequences were subsequently compared to those in the GenBank database, which was accessed through the National Center for Biotechnology Information (NCBI) website. Comparative analysis revealed that five of the seven bacterial isolates from Leucopholis coneophora exhibited more than 95% similarity to known sequences, while one isolate exhibited 90% similarity (Table S2). For the bacterial isolates from Holotrichia serrata , eight had more than 95% similarity, and the remaining two had more than 90% similarity (Table S3). Phylogenetic Tree Analysis Phylogenetic analysis of the 16S rRNA sequences revealed that the bacteria colonizing the gut of Leucopholis coneophora formed two distinct major clades with diverse subgroups. The dominant phyla in the phylogenetic tree were Firmicutes and γ-Proteobacteria, constituting the major clades. The Firmicutes clade was represented by a single genus, Bacillus , with three species and various subbranches, possibly indicating different strains. The second major clade consisted of γ-proteobacteria, represented by Klebsiella sp. and Citrobacter farmeri . The major clades of the phylogenetic tree were analyzed using 1000 bootstrap replications, with Acidobacterium capsulatum serving as the outgroup. The sequence for the outgroup was retrieved from the NCBI database. A phylogenetic tree of the seven identified gut bacteria in L. coneophora is shown in Fig. 3. A similar phylogenetic tree was constructed for the gut bacteria of Holotrichia serrata , revealing two distinct clades representing different groups: γ-proteobacteria and β-proteobacteria. The major γ-proteobacteria clade comprised the genera Pseudomonas , Citrobacter , Enterobacter , and Acinetobacter . The minor β-proteobacteria clade contained only the genus Achromobacter . This tree was also constructed with an outgroup, and the sequence was retrieved from the NCBI database. Both clades of the phylogenetic tree, along with the outgroup ( Aquifex aeolicus ), are depicted in Fig. 3, with bootstrap values based on 1000 replicates. Overall, the taxonomic classification of gut bacteria from L. coneophora and H. serrata provides valuable insights into the diversity of bacterial species present in these insects (Tables S4 & S5). Cellulolytic indices of the bacterial isolates The cellulolytic activity of the bacterial isolates was evaluated using the Congo red overlay method, where the presence of a halo zone on CMC agar plates (Figure 4B) indicated the cellulolytic index. Of the seventeen bacterial isolates tested, thirteen exhibited significant cellulolytic activity. The cellulolytic index ranged from a maximum of 1.93 ± 0.037 for the LC3B1 isolate ( Bacillus toyonensis ) to a minimum of 0.24 ± 0.015 for the LC2B3 isolate ( Klebsiella oxytoca ) from Leucopholis coneophora . Among the Holotrichia serrata strains , the highest cellulolytic index was 1.49 ± 0.04 for the H4B3 isolate ( Enterobacter sp.), while the lowest index was 0.26 ± 0.012 for H4B2 ( Enterobacter ludwigii ). The cellulolytic indices of the gut bacteria isolates are presented in Fig. 4A (Table S6). The cellulolytic indices varied significantly among the different bacterial strains, as indicated by the ANOVA results: F(2,12)=661.1, p<2×10−. Duncan’s test further confirmed these differences, revealing significant variation in the cellulolytic indices between the strains. Strain LC3B1 exhibited the highest cellulolytic index, followed by strain LC1B1. Based on these findings, four bacterial isolates—LC1B1, LC3B1, H4B3, and H5B2—were selected for further study due to their promising cellulolytic activities (Fig. 5). Substrate Degradation Ratio of Agricultural Residues The four selected bacterial isolates were cultured in media containing powdered corncob, paddy straw, and groundnut husk substrates, followed by incubation for eight days. Among the isolates, H5B2 exhibited the highest degradation efficiency, reaching 48.15 ± 1.56% for groundnut husk, whereas LC3B1 exhibited 46 ± 0.608% for paddy straw powder and 43±0.27% for corncob. The substrate degradation ratio was significantly different between the F(2, 3)= 12.236 strain (p <0.05) and the F(2,2)=8.050 strain (p<0.05) but not between substrates. The other isolates also demonstrated significant degradation capabilities, as shown in Fig. 5 (Table S7). Cellulase (β-1,4-Endoglucanase) Enzyme Assay Cellulase (β-1,4-endoglucanase) activity assays were conducted using the DNS method at 50°C and pH 4. β-1,4-endoglucanase was statistically significant among the various substrates F(2, 2)= 7.3, p <0.024 but not between strains of bacteria F(2,3)=1.16, p=0.39). Among the tested genes, LC3B1 had the highest beta 1,4-endoglucanase activity on ground nut husk, followed by LC1B1, which had the second highest endoglucanase activity (3.614 ± 0.02 U/mL), particularly on the corn cob powder substrate. LC1B1 also exhibited significant activity on groundnut husk (2.168 ± 0.07 U/mL), highlighting its potential for enzyme production. Isolates H4B3 (2.4 ± 0.03 U/mL) and H5B2 (1.626 ± 0.01 U/mL) also exhibited notable endoglucanase activities on groundnut husk. Additionally, LC1B1 exhibited activity (1.628 ± 0.01 U/mL) on paddy straw, whereas the remaining three bacteria displayed activities less than 0.7 U/mL on all three substrates. Moreover, LC3B1 exhibited considerable endoglucanase activity (2.082 ± 0.02 U/mL) on corncob. These findings underscore the substrate-specific cellulase activities of the bacterial isolates, with LC3B1 showing significant potential for enzyme production, particularly on groundnut husk and corncob substrates. The results are depicted in Fig. 6 (Table S8). FTIR and SEM Results for the Breakdown of Cellulosic Bonds in Filter Paper Structural analysis of the four bacterial isolates was performed using attenuated total reflectance (ATR) mode in the infrared range of 400–4000 cm−1, enabling the detection of bond types and functional groups (Fig. 7). The peak at 1160 cm−1 is attributed to C-O-C stretching in the cellulose/hemicellulose molecules. The peak between 2896 and 2900 cm−1 indicates C-H (β-glycosidic bond) deformation in the polysaccharide molecules. The stretching of carbon and oxygen molecules between 1749 and 1599 cm−1 revealed decomposition products such as ketones, aldehydes, alcohols, and carboxylic acids derived from cellulose and hemicellulose. Additionally, the shifts in the O–H and C–H stretching vibrations at 3333 and 2894 cm−1 can be attributed to changes in the intra- and intermolecular bonds of the cellulosic polymer. The FTIR results revealed broad absorption bands at 897, 1651, and 2900 cm−1, corresponding to COC, CCO, OCH deformation, C-5, C-6 motion stretching, OH bending, and CH stretching, respectively. The intense peak at 3000–3700 cm−1 is attributed to the stretching of OH functional groups and amine groups. Field emission scanning electron microscopy (FESEM) provided insights into the significant structural changes in the surface morphology of both the control and treated filter paper (FP) samples (Fig. 8). Compared to those of the control substrates, the treated FP surface exhibited a rough appearance, likely due to bacterial adherence and superficial hydrolysis of the biomass. The bacterial cells adhered to the substrate, potentially creating pores and tunnels that allowed access to the internal fibers of the cellulosic biomass. This process suggested that the long cellulose chains in the FPs released their microfibers. The electron micrographs further demonstrated substrate disruption, as evidenced by the accumulation of hydrolyzed biomass on the surface. Discussion Insects host a diverse array of microorganisms in their guts, which play vital roles in fulfilling the insects' physiological and ecological needs. These microorganisms enable insects to survive in harsh environmental conditions by aiding in digestion and the assimilation of food. Moreover, gut bacteria detoxify harmful substances and protect insects from pathogens by producing antimicrobial compounds (Jang and Kikuchi, 2020) [ 46 ]. Lemke et al. (2003) [ 14 ] conducted a comprehensive study on the microbiota of the white grub species Pachnoda ephippiata , finding that both the hindgut and midgut harbored diverse microorganisms involved in cellulose degradation, microbial fermentation, and proteolytic activities. Although numerous studies have reported the diversity and functional roles of bacteria in the guts of various white grub species [ 47 – 52 ], no reports exist on the bacterial diversity in H. serrata and L. coneophora , which are native to Karnataka, India. In this study, 17 bacterial isolates were obtained from the fermentation chambers of L. coneophora and H. serrata . Molecular characterization through 16S rRNA gene sequencing revealed significant diversity among the isolated bacterial strains, with the most dominant families being Enterobacteriaceae, followed by Bacillaceae and Pseudomonadaceae, aligning with previous findings in other white grub species [ 53 – 55 ]. Phylogenetic analysis further elucidated the evolutionary relationships among these isolates, revealing the presence of diverse taxa such as Firmicutes and Proteobacteria, consistent with other studies on white grub species [ 16 , 35 ]. The cellulolytic activity of these bacteria was assessed by measuring the zone of clearance surrounding bacterial colonies, indicative of carboxymethyl cellulose (CMC) hydrolysis by secreted cellulase (CMCase). Thirteen of the isolated bacteria exhibited significant cellulolytic activity. The isolate LC3B1 ( Bacillus toyonensis ) from L. coneophora had the highest cellulolytic index (1.93 ± 0.037), while H4B3 ( Enterobacter sp.) from H. serrata showed the highest cellulolytic index (1.49 ± 0.04). Additionally, substrate degradation abilities of selected bacterial strains demonstrated promising results, consistent with earlier reports on bacterial isolates from other white grub species [ 58 – 60 ]. Pretreatment of substrates before degradation was found to enhance degradation efficiency, with bacterial isolates from rumen showing higher efficiency than gut bacteria [ 35 , 61 ]. Cellulase enzyme assays highlighted distinct substrate-specific activities among the bacterial isolates, emphasizing their potential for lignocellulosic biomass conversion [ 59 ]. For instance, Klebsiella pneumoniae , Klebsiella sp., and Bacillus sp. have been identified as significant cellulase producers in studies on bacterial cellulase from insect guts, with Klebsiella pneumoniae exhibiting endoglucanase activity at 3.5 U/mL [ 62 ] and Klebsiella variicola showing 0.092 U/mL cellulolytic activity [ 63 ]. In another study, cellulose-degrading bacteria from invertebrates such as bookworms, termites, snails, and caterpillars exhibited extracellular cellulase activities ranging from 0.012 to 0.196 IU/mL for filter paper cellulase (FPC) and from 0.162 to 0.400 IU/mL for endoglucanase assay [ 54 ]. The β-1,4-endoglucanase enzyme assay, as a preliminary test, is noteworthy for optimizing enzyme production under varying pH values, temperatures, substrates, and metal ion concentrations. Further insights into the breakdown of cellulosic bonds were obtained using FTIR and SEM, which confirmed the enzymatic destruction of cellulose by the isolated strains [ 43 , 64 ]. Functional group absorbance bands associated with structural changes in biomass were attributed to C-O, C = C, and C-C-O stretching at 1035 cm⁻¹, C-H stretching at 2840 cm⁻¹ and 2937 cm⁻¹, O-H in-plane bending at 1440 cm⁻¹, and C = O stretching (unconjugated) at 1682 cm⁻¹, present in cellulose, hemicellulose, and lignin [ 44 , 45 , 65 ]. In conclusion, this study is the first to identify cellulolytic bacteria from the gut microbiota of the white grub species H. serrata and L. coneophora . These bacteria exhibit significant potential for the degradation of complex, cellulose-rich organic wastes. The prevalence of cellulase-producing strains from families such as Enterobacteriaceae, Bacillaceae, and Pseudomonadaceae highlights their crucial role in lignocellulosic biomass degradation. The variability in cellulose degradation efficiency across different bacterial strains and substrates underscores the substrate-specific nature of cellulose breakdown. The cellulose degradation process yields reducing sugars like glucose, which are essential for bioethanol production. Utilizing these reducing sugars can reduce dependence on traditional raw materials like sugar and corn, which are commonly used in conventional ethanol production. Effective lignocellulose degradation thus requires the application of efficient microbial strains. Consequently, these findings underscore the potential for sustainable waste management and biofuel production through bioconversion techniques that leverage cellulolytic bacteria isolated from white grub guts. This approach could significantly enhance the sustainability and value of biofuel production. Declarations Acknowledgments: RBN acknowledges the Science and Engineering Research Board, New Delhi for the Fund TAR/2020/000014. RBN acknowledges ICAR-CIPHET and Ludhiana for providing support. RBN thanks T. Govindaraju of JNCASR for supporting the FTIR and SEM studies. A. Ethics approval and consent to participate The insects were predominantly pestiferous and were collected from farmer fields after providing consent. B. Consent for publication Not applicable C. Availability of data and materials The data were submitted to NCBI under accession numbers STRAIN ID ACCESSION NUMBER SPECIES CLOSEST STRAIN IN GENBANK IDENTITY (%) Leucophalis coneophora CDBLC1B1 PP542502 Klebsiella sp. Klebsiella sp. HaMC2 99.50 CDBLC1B2 PP542506 Bacillus siralis Bacillus siralis J28TS8 99.12 CDBLC2B1 PP542505 Bacillus toyonensis Bacillus toyonensis strain MB14 90 CDBLC2B2 PP542504 Bacillus cereus Bacillus cereus strain ABC10 95 CDBLC2B3 PP542497 Klebsiella oxytoca Klebsiella oxytoca strain S1-2-2 98.21 CDBLC3B1 PP542499 Citrobacter farmeri Citrobacter farmeri isolate CCB19B 97.7 CDBLC6B1 PP542500 Klebsiella pasteurii Klebsiella pasteurii strain SPARK1448C2 98.28 Holotrichia serrata CDBH2B1 PP542512 Pseudomonas sp. Pseudomonas sp.strain H374 99.13 CDBH3B1 PP542515 Pseudomonas monteilii Pseudomonas monteilii 99.14 CDBH3B2 PP542516 Pseudomonas urethralis Pseudomonas urethralis strain BML-PP042 99.07 CDBH3B3 PP542518 Achromobacter piechaudii Achromobacter piechaudii strain ERG1 93.38 CDBH3B4 PP542520 Citrobacter koseri Citrobacter koseri strain IHB B 6842 98.49 CDBH4B1 PP542525 Enterobacter tabaci Enterobacter tabaci strain TBMAX92 96.49 CDBH4B2 PP542528 Enterobacter ludwigii Enterobacter ludwigii strain AA1 96.56 CDBH4B3 PP542530 Enterobacter sp. Enterobacter sp.JBIWA003 97.68 CDBH5B1 PP542527 Enterobacter sp. Enterobacter sp.strain LFR1 99.1 CDBH5B2 PP542526 Acinetobacter baumannii Acinetobacter baumannii strain JC359 90 D. Competing Interests There are no competing interests. E. Funding This work was funded by the Science and Engineering Research Board under Teacher Associateship Research Excellence, TAR/2020/000014 E. Authors' contributions RBN and PKV: conceptualization of work GVV- Conducted the experiment and collected the data PKV- and GVV-Collected insects for the study PKV-Identification of white grubs and dissection of the fermentation chamber RBN- Supervised overall study and edited the manuscript BP- Corrected the manuscript GKR- Involved in collecting the experimental data References Steinhaus EA. A study of the bacteria associated with thirty species of insects. J Bacteriology. 1941; 42:757-790. Ladouceur EE, Wood SC, Laudier D, Simko E. Arthropoda: insecta. Invertebrate Histology. 2021;301-317. Engel P, Moran NA. The gut microbiota of insects–diversity in structure and function. FEMS Microbiol Rev. 2013;37(5):699-735. Jing TZ, Qi FH, Wang ZY. Most dominant roles of insect gut bacteria: digestion, detoxification, or essential nutrient provision?. Microbiome. 2020;8(1):1-20. Xie SX, Syrenne R, Sun S, Yuan JS. 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The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res Spec Publ. 1997;25(24): 4876-4882. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;16:111-120. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):547. Teather RM, Wood PJ, Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. AEM. 1982;43(4): 777-780. Handique G, Phukan A, Bhattacharyya B, Baruah AAL H, Rahman SW, Baruah R. Characterization of cellulose degrading bacteria from the larval gut of the white grub beetle Lepidiota mansueta (Coleoptera: Scarabaeidae). AIBPEA. 2017;94(2),:21370. Updegraff DM. Semimicro determination of cellulose inbiological materials. Anal biochem. 1969;32(3):420-424. Dar MA, Syed R, Pawar KD, Dhole NP, Xie R, Pandit RS, Sun J. Evaluation and characterization of the cellulolytic bacterium, Bacillus pumilus SL8 isolated from the gut of oriental leafworm Spodoptera litura : An assessment of its potential value for lignocellulose bioconversion. Environ Technol Innov. 2022; 27:102459. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal chem. 1959;31(3):426-428. Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59(2): 257-268. Prasad RK, Chatterjee S, Mazumder PB, Sharma S, Datta S, Vairale M. G, Dwivedi SK. Study on cellulase (Β-1,4-endoglucanase) activity of gut bacteria of Sitophilus oryzae in cellulosic waste biodegradation. Bioresour Technol. 2019;7:100274. Sheoran OP, Tonk DS, Kaushik LS, Hasija RC, Pannu RS. Statistical software package for agricultural research workers. Recent advances in information theory, statistics & computer applications by DS Hooda & RC Hasija Department of Mathematics Statistics,CCS HAU Hisar. 1998;8(12):139-143. Lee SH, H'ng PS, Lee AN, Sajap AS, Tey BT, Salmiah U. Production of pyroligneous acid from lignocellulosic biomass and their effectiveness against biological attacks. J Appl Sci. 2010;10(20): 2440-2446. Lee CM, Kubicki JD, Fan B, Zhong L, Jarvis MC, Kim SH. Hydrogen-bonding network and OH stretch vibration of cellulose: comparison of computational modeling with polarized IR and SFG spectra. JPC B. 2015;119(49):15138-15149. Sharma S, Sharma V, Kuila A. Cellulase production using natural medium and its application on enzymatic hydrolysis of thermo chemically pretreated biomass. 3 Biotech. 2016;6: 1-11. Jang S, Kikuchi Y. Impact of the insect gut microbiota on ecology, evolution, and industry.Curr Opin Insect Sci. 2020;41:33-39 Cazemier AE, Verdoes JC, Reubsaet FA, Hackstein JH, van der Drift C, Op den Camp HJ. Promicromonospora pachnodae sp. nov., a member of the (hemi) cellulolytic hindgut flora of larvae of the scarab beetle Pachnoda marginata . Antonie van Leeuwenhoek.2003; 83 (2): 135–148. Zhang H, Jackson TA. Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae). J. Appl. Microbiol. 2008;105 (5):1277–1285. Egert M, Stingl U, Dyhrberg Bruun L, Pommerenke B, Brune A, Friedrich MW. Structure and topology of microbial communities in the major gut compartments of Melolontha melolontha larvae (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 2005;71 (8): 4556–4566 Handique G, Phukan A, Bhattacharyya B, Baruah AALH, Rahman SW, Baruah R. Characterization of cellulose degrading bacteria from the larval gut of the white grub beetle Lepidiota mansueta (Coleoptera: Scarabaeidae). Arch. Insect Biochem. Physiol.2017; 94 (2): 21370. Surabhi K, Rangeshwaran R, Frenita ML, Shylesha AN, Jagadeesh P. Isolation and characterization of the culturable microbes associated with gut of adult dung beetle Onitis philemon (Fabricius). J. Pharmacogn. Phytochem. 2018;27: 1609–1614 MsangoSoko K, Chandel R, Gandotra S, Yadav K, Gambhir S, Subramanian KSS. Diversity of microbial groups associated with the gut of the eri silkworm, Samia ricini , (Lepidoptera: Saturniidae) and white grub, Anomala dimidiata, (Coleoptera: Scarabaeidae) larvae as revealed by phospholipid fatty acids. J. Entomol. Zool. Stud.2020; 8 (2): 1679–1683. Banerjee S, Maiti TK, Roy RN. Production, purification, and characterization of cellulase from Acinetobacter junii GAC 16.2, a novel cellulolytic gut isolate of Gryllotalpa africana, and its effects on cotton fiber and sawdust. Ann microbiol. 2020;70(1); 1-16. Goswami K, DekaBoruah HP, Saikia R. Production of cellulase by Novosphingobium sp. Cm1 and its potential application in lignocellulosic waste hydrolysis. Prep biochem biotech. 2022;52(6):724-735. Gupta P, Samant K, Sahu A. Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential. Int J Microbiol.2012;1:578925. Prem Anand AA, Vennison SJ, Sankar SG, Gilwax Prabhu DI, Vasan PT, Raghuraman T, Vendan SE. Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion. J Insect Sci. 2010;10(1):107. Danu N, Paschapur A, Subbanna ARNS, Stanley J, Singh AK, Bisht I, Gupta JP. Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas. J Asia Pac Entomol. 2023;26(1):102036. Wakita Y, Shimomura Y, Kitada Y, Yamamoto H, Ohashi Y, Matsumoto M. Taxonomic classification for microbiome analysis, which correlates well with the metabolite milieu of the gut. BMC microbiol.2018;18: 1-11. Hankin L, Anagnostakis SL. Solid media containing carboxymethylcellulose to detect Cx cellulase activity of microorganisms. Microbiol.1977;98(1): 109-115. Schwarz W. The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol. 2001;56: 634-649. Pourramezan Z, Ghezelbash GR, Romani B, Ziaei S, Hedayatkhah A. Screening and identification of newly isolated cellulose-degrading bacteria from the gut of xylophagous termite Microcerotermes diversus (Silvestri). Microbiol. 2012;81: 736-742. Rajeswari G, Jacob S, Chandel AK, Kumar V. Unlocking the potential of insect and ruminant host symbionts for recycling of lignocellulosic carbon with a biorefinery approach: a review. Microb cell fact. 2021;20(1):107. Barbosa KL, dos Santos Malta VR, Machado SS, Junior GAL, da Silva AP V, Almeida RMRG, da Luz JMR. Bacterial cellulase from the intestinal tract of the sugarcane borer. Int J Biol Macromol. 2020;161: 441-448. Gopinath S M, Shareef I, Ashalatha Ranjit S. Isolation, screening and purification of cellulase from cellulase producing Klebsiella variicola RBER3 (KF036184. 1). Int J Sci Res. 2012;3: 2319-7064. Hospodarova, V., Singovszka, E, Stevulova, N. Characterization of cellulosic fibers by FTIR spectroscopy for their further implementation to building materials. AJAC. 2018;9(6), 303-310 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile2452024.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4958316","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":352544062,"identity":"526da4a2-e9bd-4620-89c9-f574b41d8106","order_by":0,"name":"Gatta Vis","email":"","orcid":"","institution":"Department of Plant Biotechnology, UAS, GKVK, Bangalore","correspondingAuthor":false,"prefix":"","firstName":"Gatta","middleName":"","lastName":"Vis","suffix":""},{"id":352544063,"identity":"5a67a533-3a9e-4e16-aed2-69866aae6a04","order_by":1,"name":"KV Prakash","email":"","orcid":"","institution":"ICAR-AINP on Soil Arthropod Pests, UAS, GKVK","correspondingAuthor":false,"prefix":"","firstName":"KV","middleName":"","lastName":"Prakash","suffix":""},{"id":352544064,"identity":"c42de5f1-9bea-4287-acfe-594215e80412","order_by":2,"name":"BN Ramesh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYLACxoYDDPzMzAeATAkZ4rVItrclgLTwEK/F4MwZAxCbsBbz9tOJD3/uuJPHcCPn86sbNRY8DOyHj27Ap0XmTO5mY94zz4oZZ+Rus845BnQYT1raDXxaJBhyt0kzth1ObJbI3WacwwbUIsFjhl8L/9vtP38CtbRJ5DwzzvlHjBag4Qy8QC09PGeYH+e2EaXl7WZp3rZniTPY28yYc/skeNgI+oU/d+PHn213EvcfZn78OedbnRw/++FjeLUgAzYJMEmschBg/kCK6lEwCkbBKBg5AAAGaE0VDOkYdQAAAABJRU5ErkJggg==","orcid":"","institution":"ICAR-AICRP On Post Harvest Engineering and Technology, University of Agricultural Sciences, Bangalore, India","correspondingAuthor":true,"prefix":"","firstName":"BN","middleName":"","lastName":"Ramesh","suffix":""},{"id":352544066,"identity":"892d0fc3-7898-447e-b4f4-cace4aececf8","order_by":3,"name":"P Bhavani","email":"","orcid":"","institution":"Department of Plant Biotechnology, UAS, GKVK, Bangalore","correspondingAuthor":false,"prefix":"","firstName":"P","middleName":"","lastName":"Bhavani","suffix":""},{"id":352544068,"identity":"d97a8a33-f6a3-44ef-bec8-1ec28f2f5ea1","order_by":4,"name":"KR Gagandeep","email":"","orcid":"","institution":"Department of Plant Biotechnology, UAS, GKVK, Bangalore","correspondingAuthor":false,"prefix":"","firstName":"KR","middleName":"","lastName":"Gagandeep","suffix":""}],"badges":[],"createdAt":"2024-08-22 13:16:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4958316/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4958316/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65068308,"identity":"54240135-905b-47e2-9e47-80d0a28219b4","added_by":"auto","created_at":"2024-09-23 09:21:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1185915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eHolotrichia serrata \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeucopholis coneophora \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(B)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/3b84bf00bf132b67865912c2.png"},{"id":65068311,"identity":"b5e80280-ecf0-4f13-ad69-614c86df4aeb","added_by":"auto","created_at":"2024-09-23 09:21:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative fermentation chambers isolated from the two white grubs used in the present study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/7bb1aeb508284d2316113202.png"},{"id":65069586,"identity":"86b2538a-8729-4bed-a8d8-d6e671f548c6","added_by":"auto","created_at":"2024-09-23 09:37:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree showing the evolutionary relationships of the bacterial isolates from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolotrichia serrata\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/cf66399cdd4280c2e6bfc714.png"},{"id":65068826,"identity":"b1d1b64c-b112-4e2b-a723-eb6f7556d090","added_by":"auto","created_at":"2024-09-23 09:29:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":452559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA Cellulolytic indices of gut bacteria isolated from two white grub speciesL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: Cellulolytic indices of gut bacteria isolated from two white grub species (a-LC1B1, b-LC3B1, c-H4B3, and d-H5B2)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/a42baec72373c0a14c75ecca.png"},{"id":65069587,"identity":"0ce28f5d-e56a-411c-9430-9a06c60950f3","added_by":"auto","created_at":"2024-09-23 09:37:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate degradation ratio (%) of cellulolytic isolates with different agricultural residues\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/30be2f4022d50733a45d6281.png"},{"id":65068828,"identity":"cbbf8de7-fad9-4520-a5da-6d34fc2b0403","added_by":"auto","created_at":"2024-09-23 09:29:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":85665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndoglucanase activity (U/mL) of cellulolytic isolates with different agricultural substrates\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/82445e16e191cf8484f3e63d.png"},{"id":65068316,"identity":"4da0441a-7c34-4bce-a915-1154a2e16443","added_by":"auto","created_at":"2024-09-23 09:21:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":256199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR profiles of the control and filteredfilter paper treated with the LC1B1, LC3B1, H4B3, and H5B2 bacterial isolates\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/611902e52203a72f81a9b444.png"},{"id":65068317,"identity":"95971313-1943-49f4-a6fa-2ac1b1ceead5","added_by":"auto","created_at":"2024-09-23 09:21:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":833720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectron micrograph showing the adhesion of cells and degradation of the filter paper due to bacteria (A-LC1B1-, B-LC3B1, C-CONTROL, D-H4B3 and E-H5B2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/0db95687cdedaec735688b18.png"},{"id":65071171,"identity":"6b43a69c-e406-462f-b85d-5102027032b1","added_by":"auto","created_at":"2024-09-23 09:53:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5935903,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/b09ddda8-902b-4a03-b02d-a7e3da434bf0.pdf"},{"id":65068315,"identity":"54d58fea-6094-4827-9922-79970da76b6a","added_by":"auto","created_at":"2024-09-23 09:21:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2384341,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2452024.docx","url":"https://assets-eu.researchsquare.com/files/rs-4958316/v1/edcb38bea3c9bddf398ac76e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Cellulolytic Gut Bacteria Isolated from White Grubs (Holotrichia serrata and Leucopholis coneophora) and Their Utilization in Lignocellulose Degradation","fulltext":[{"header":"Background","content":"\u003cp\u003eInsects are among the most omnipresent and dominant organisms on Earth, constituting the largest and most diversified class within the phylum Arthropoda. This extensive diversity makes Insecta a significant and widespread group within the animal kingdom, characterized by their remarkable adaptability and ecological success. A critical aspect of insect biology is their symbiotic interactions with microorganisms, which range from mutualistic to parasitic relationships. Among these, the insect gut microbiota plays a pivotal role in aiding digestion and nutrient acquisition, forming complex and varied colonies of bacteria, fungi, and protozoa within the digestive system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese symbiotic microorganisms are particularly important for the digestion of lignocellulosic biomass, a primary component of the plant cell wall, which many insects utilize as a food source. Lignocellulose, composed mainly of cellulose, hemicellulose, and lignin, is notoriously difficult to degrade due to its complex structure. Insects, however, have evolved highly specialized digestive systems, often complemented by their gut microbiota, to break down these complex carbohydrates. Notably, termites and certain beetles are known to harbor gut bacteria from the phyla Firmicutes and Proteobacteria, which facilitate cellulose degradation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The gut microflora performs many functions, including synthesizing novel compounds and enzymes with therapeutic and industrial applications [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, microbiota is known to contribute to bioremediation and the degradation of plastics [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], as well as playing roles in disease prevention and pest management [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite extensive research on termites and beetles, there is a notable gap in understanding the cellulose-degrading capabilities of white grubs.\u003c/p\u003e \u003cp\u003eWhite grubs, the larvae of scarab beetles, are polyphagous subterranean herbivores that pose significant agricultural challenges. The two white grub species, \u003cem\u003eHolotrichia serrata\u003c/em\u003e and \u003cem\u003eLeucopholis coneophora\u003c/em\u003e, which are major pests in Karnataka, India are the major focus of this study. These species are of particular interest due to their ability to feed on the roots of economically important crops, such as sugarcane and arecanut, which are rich in cellulose and nitrogenous compounds [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The present investigation aims to isolate and characterize the gut microbiota of these white grub species, with a specific focus on their potential to degrade lignocellulose. Understanding the symbiotic relationships between these insects and their gut microbiota not only contributes to the basic understanding of pest biology and host-microbiome co-evolution but also highlights the potential applications of these findings in bioengineering digestive enzymes for industrial use, including in biorefinery and biofuel production [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubstrate Preparation\u003c/h2\u003e \u003cp\u003eAgricultural wastes such as paddy straw, groundnut husk, and corncob powder were collected from local fields at the University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bengaluru, Karnataka, India. The pretreatment of these agricultural wastes involved treating them separately with a mild alkaline solution (0.1 N NaOH, w/v). Following pretreatment, the samples were thoroughly washed with deionized water to achieve a neutral pH, air-dried, finely milled, and then sieved through a 2.0 mm screen to ensure uniform particle size. The pretreated substrates were either used immediately for hydrolysis experiments or stored in airtight containers for future use [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInsect sample collection\u003c/h2\u003e \u003cp\u003eThe third-instar larvae of two white grub species, \u003cem\u003eLeucopholis coneophora\u003c/em\u003e and \u003cem\u003eHolotrichia serrata\u003c/em\u003e, were collected from infested fields in Moodabidri, Dakshina Kannada district, and Mahadeshwarapura, Mandya district of Karnataka, India, respectively. The identification of these white grub species was performed by Dr. K.V. Prakash, an entomologist, using binomial keys. The larvae were transported to the laboratory in sterilized, aerated plastic containers and were starved for 24 hours before dissection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of Gut Bacteria\u003c/h2\u003e \u003cp\u003eThe larvae were rinsed in double distilled water for 30 seconds, followed by 70% ethanol for 60 seconds, and then rinsed again in double distilled water for 30 seconds to remove the disinfectant. The sterilized larvae were then dissected under laminar airflow using sterile microscissors to extract the gut. The fermentation chamber was isolated from the gut and carefully removed [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe dissected fermentation chamber was pooled into a 1.5 mL microtube containing 1.0 mL of phosphate-buffered saline (PBS, pH 7.4) and macerated using a sterile micropestle. The homogenized gut extracts were then enriched in Berg Minimal Salt media (BMS), which was supplemented with 2 g/L NaNO₃, 0.02 g/L MgSO₄\u0026middot;7H₂O, 0.02 g/L MnSO₄\u0026middot;H₂O, 0.5 g/L K₂HPO₄, 0.02 g/L FeSO₄\u0026middot;7H₂O, and 0.5 g/L CaCl₂\u0026middot;2H₂O, containing 1% (w/v) carboxymethyl cellulose (CMC) as the substrate [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The seeded medium was incubated at 37\u0026deg;C with shaking at 150 rpm for 48 hours. Following incubation, the culture broth was serially diluted in PBS (pH 7.4) to a final dilution of 10⁻⁷. One hundred microliters of each dilution were spread onto BMS-CMC agar plates and incubated at 37\u0026deg;C for 48 hours. Colonies were purified by repeated streaking on LB agar media, and the isolated strains were stored at -80\u0026deg;C in glycerol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Characterization and Identification of Isolated Bacterial Strains:\u003c/h2\u003e \u003cp\u003eGenomic DNA from the bacterial isolates was extracted following a modified protocol [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The bacterial isolates were cultured in LB broth and incubated overnight at 37\u0026deg;C with shaking. Approximately 1.5 mL of each culture was transferred to a microcentrifuge tube and centrifuged for 7 minutes. The supernatant was carefully removed, and the pellet was resuspended in TE buffer containing 100 mg/mL lysozyme, 20 mg/mL proteinase K, and 10% SDS. This mixture was incubated for one hour at 37\u0026deg;C. Following incubation, 5 M NaCl and CTAB solution were added, and the mixture was incubated at 65\u0026deg;C for 10 minutes. The sample was then washed with a mixture of chloroform and isoamyl alcohol (24:1), and the aqueous phase was transferred to a fresh tube. An equal volume of phenol:chloroform: isoamyl alcohol (25:24:1) was added, followed by centrifugation at 8,000 rpm for 5 minutes at 4\u0026deg;C. This step was repeated until a clear supernatant was obtained. DNA precipitation was achieved by adding an equal volume of chilled isopropanol, mixing gently, and incubating overnight at -20\u0026deg;C. The DNA was pelleted by centrifugation at 10,000 rpm for 20 minutes at 4\u0026deg;C, washed with 70% ethanol, air-dried, and finally dissolved in TE buffer.\u003c/p\u003e \u003cp\u003ePCR amplification of the 16S rRNA gene was performed using the specific primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The thermal cycling conditions were as follows: an initial denaturation at 95\u0026deg;C for 5 minutes, followed by 35 cycles of denaturation at 95\u0026deg;C for 30 seconds, annealing at 55\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 1 minute, with a final extension at 72\u0026deg;C for 7 minutes. The PCR products were assessed for size and purity on 1.5% (w/v) agarose gels, after which they were purified and sequenced. The resulting partial sequences were aligned using the BioEdit tool [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. BLAST analysis was conducted to identify closely related species in the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The sequences were further aligned using the ClustalW multiple sequence alignment tool [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and a phylogenetic tree was constructed using the neighbor-joining method with the Kimura 2 evolutionary model [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The tree construction included a bootstrap value of 1000 replications and was performed using MEGA 11 software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eScreening of cellulose-degrading bacteria\u003c/h2\u003e \u003cp\u003eThe bacterial isolates were inoculated onto BMS agar plates supplemented with carboxymethylcellulose (CMC) as the substrate and incubated at 37\u0026deg;C for 48 hours. To assess CMC degradation by the bacterial isolates, the plates were flooded with 0.1% Congo red solution and allowed to stain for 30 minutes. Excess Congo red was then removed, and the plates were treated with 1 M NaCl solution for 15 minutes. Degradation of the β-1,4 glycosidic bonds by cellulase activity prevented Congo red from binding to the cellulose polymer, resulting in clear zones on the medium [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBacterial colonies that exhibited the most distinct and largest diameter zones of hydrolysis on the stained plates were selected for further investigations. The cellulolytic indices of the isolates were measured following a previously published protocol with slight modifications [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCellulolytic index = (diameter of zone of clearance\u0026thinsp;\u0026minus;\u0026thinsp;diameter of bacterial colony)/ diameter of bacterial colony.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSubstrate Degradation Ratio\u003c/h2\u003e \u003cp\u003eBriefly, 1 mL of freshly grown bacterial culture (OD600: 0.5) was inoculated into BMS media supplemented with different substrates, followed by incubation in a rotary shaker at 37\u0026deg;C and 150 rpm for 7 days. After incubation, the mixture was centrifuged at 10,000 rpm for 15 minutes at 4\u0026deg;C, and the supernatant was collected as a crude enzyme extract, which was stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo prepare the substrates, they were washed with 3 mL of acetic nitric reagent (a mixture of 150 mL 80% acetic acid, and 15 mL concentrated nitric acid). The substrates were then washed with distilled water for 5 minutes, followed by a wash with absolute ethanol for 10 minutes. The residues were subsequently dried at 60\u0026deg;C in a hot air oven [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The final weight of the substrates was \u003cb\u003emeasured\u003c/b\u003e, and the percentage of degradation was calculated using the following formula from Updegraff, 1969 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSubstrate Degradation (%) = {(Initial weight of substrate\u0026thinsp;\u0026minus;\u0026thinsp;Final weight of substrate) / Initial weight of substrate} \u0026times;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCellulase (β-1,4-Endoglucanase) Enzyme Assay\u003c/h2\u003e \u003cp\u003eThe supernatant obtained from the previous experiment was used as the crude enzyme. For the enzyme assay, 0.5 mL of the supernatant was mixed with 1 mL of 0.05 M citrate buffer (pH 4.5) containing 1% CMC as the substrate. The reaction mixture was incubated in a water bath for 60 minutes. After incubation, 3 mL of DNS (dinitro salicylic acid) reagent was added to each tube containing the reaction mixture, which was then heated at 100\u0026deg;C for 5 minutes and subsequently cooled at 4\u0026deg;C for 5 minutes to stop the enzymatic reaction [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe absorbance of the reaction mixture was measured at 540 nm using a spectrophotometer, with a control sample as the baseline. The amount of reducing sugars produced was determined using a glucose standard. Enzymatic activity was expressed in units (U/mL), where one unit is defined as the amount of enzyme that releases 1 \u0026micro;mol of reducing sugars (measured as glucose) per mL per minute [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSEM Analysis of the Hydrolyzed Substrate\u003c/h2\u003e \u003cp\u003eThe hydrolyzed and control substrates were characterized using Fourier-transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). A 1% bacterial inoculum was added to freshly prepared BMS liquid media containing 1000 mg of filter paper (FP) as the substrate, and the culture was incubated for 14 days at 150 rpm and 37\u0026deg;C. A control experiment was conducted under the same conditions but without bacterial inoculation.\u003c/p\u003e \u003cp\u003eAfter the 14-day incubation period, the broth was centrifuged at 5000 rpm for 5 minutes to extract the biomass. For FESEM analysis, the samples were prepared following a previously published protocol with slight modifications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The filter paper was washed with distilled water and dried overnight at 60\u0026deg;C. The samples were then sputter-coated with a 100 \u0026Aring; layer of gold in an argon gas atmosphere to enhance conductivity and reduce charging effects during scanning electron microscopy analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFTIR Analysis of the Hydrolyzed Substrate\u003c/h2\u003e \u003cp\u003eFourier-transform infrared (FTIR) spectroscopy is a rapid and nondestructive technique for detecting functional groups in the mid-IR region. In this study, FTIR spectroscopy was used to analyze filter paper, which served as the sole carbon source for bacterial growth. After 14 days of bacterial treatment, filter paper samples, along with control samples, were mixed with 1000 mg of spectroscopy-grade potassium bromide (KBr) in an agate mortar and pressed into discs.\u003c/p\u003e \u003cp\u003eThe infrared (IR) spectra were recorded using an FTIR spectrometer in transmission mode, covering the range from 400 to 4000 cm⁻\u0026sup1; [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. SEM and FTIR analyses were conducted using the instruments available at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) in Bengaluru.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe results for cellulolytic activity, substrate degradation, and enzyme assays were analyzed using descriptive statistics, analysis of variance (ANOVA), and Duncan's multiple range test (DMRT) to evaluate differences between factors at p\u0026thinsp;=\u0026thinsp;0.05. The statistical analyses were performed using OP STAT [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and the results were interpreted with Microsoft Excel (version 2021). FTIR data were plotted and analyzed using OriginPRO (version 2023b; OriginLab, Northampton, MA, USA) software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIsolation of Cellulolytic Gut Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphological characteristics of bacterial isolates 1 to 7 from \u003cem\u003eLeucopholis coneophora\u003c/em\u003e and 8 to 17 from \u003cem\u003eHolotrichia serrata\u003c/em\u003e are provided in the Supplementary Data (Table S1). In total, seventeen bacterial isolates were obtained from the fermentation chambers (Figure 2) of two white grub species collected from distinct regions. Seven isolates were derived from \u003cem\u003eL. coneophora\u003c/em\u003e, while ten were obtained from \u003cem\u003eH. serrata\u003c/em\u003e (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular characterization of cellulolytic gut bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from all the samples, and 16S rRNA gene amplification using universal 16S rRNA primers produced high-quality amplicons, with sizes ranging from approximately 1450 bp (Figures S1 and S2). These PCR products were subsequently sequenced, and the resulting sequences were aligned using the BioEdit tool. The sequences were subsequently compared to those in the GenBank database, which was accessed through the National Center for Biotechnology Information (NCBI) website.\u003c/p\u003e\n\u003cp\u003eComparative analysis revealed that five of the seven bacterial isolates from Leucopholis coneophora exhibited more than 95% similarity to known sequences, while one isolate exhibited 90% similarity (Table S2). For the bacterial isolates from \u003cem\u003eHolotrichia serrata\u003c/em\u003e, eight had more than 95% similarity, and the remaining two had more than 90% similarity (Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic Tree Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of the 16S rRNA sequences revealed that the bacteria colonizing the gut of \u003cem\u003eLeucopholis coneophora\u003c/em\u003e formed two distinct major clades with diverse subgroups. The dominant phyla in the phylogenetic tree were Firmicutes and γ-Proteobacteria, constituting the major clades. The Firmicutes clade was represented by a single genus, \u003cem\u003eBacillus\u003c/em\u003e, with three species and various subbranches, possibly indicating different strains. The second major clade consisted of γ-proteobacteria, represented by \u003cem\u003eKlebsiella\u003c/em\u003e sp. and \u003cem\u003eCitrobacter farmeri\u003c/em\u003e. The major clades of the phylogenetic tree were analyzed using 1000 bootstrap replications, with \u003cem\u003eAcidobacterium capsulatum\u003c/em\u003e serving as the outgroup. The sequence for the outgroup was retrieved from the NCBI database. A phylogenetic tree of the seven identified gut bacteria in \u003cem\u003eL. coneophora\u003c/em\u003e is shown in Fig. 3.\u003c/p\u003e\n\u003cp\u003eA similar phylogenetic tree was constructed for the gut bacteria of \u003cem\u003eHolotrichia serrata\u003c/em\u003e, revealing two distinct clades representing different groups: γ-proteobacteria and β-proteobacteria. The major γ-proteobacteria clade comprised the genera \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eCitrobacter\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, and \u003cem\u003eAcinetobacter\u003c/em\u003e. The minor β-proteobacteria clade contained only the genus \u003cem\u003eAchromobacter\u003c/em\u003e. This tree was also constructed with an outgroup, and the sequence was retrieved from the NCBI database. Both clades of the phylogenetic tree, along with the outgroup (\u003cem\u003eAquifex aeolicus\u003c/em\u003e), are depicted in Fig. 3, with bootstrap values based on 1000 replicates.\u003c/p\u003e\n\u003cp\u003eOverall, the taxonomic classification of gut bacteria from \u003cem\u003eL. coneophora\u003c/em\u003e and \u003cem\u003eH. serrata\u003c/em\u003e provides valuable insights into the diversity of bacterial species present in these insects (Tables S4 \u0026amp; S5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellulolytic indices of the bacterial isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cellulolytic activity of the bacterial isolates was evaluated using the Congo red overlay method, where the presence of a halo zone on CMC agar plates (Figure 4B) indicated the cellulolytic index. Of the seventeen bacterial isolates tested, thirteen exhibited significant cellulolytic activity. The cellulolytic index ranged from a maximum of 1.93 ± 0.037 for the LC3B1 isolate (\u003cem\u003eBacillus toyonensis\u003c/em\u003e) to a minimum of 0.24 ± 0.015 for the LC2B3 isolate (\u003cem\u003eKlebsiella oxytoca\u003c/em\u003e) from \u003cem\u003eLeucopholis coneophora\u003c/em\u003e. Among the \u003cem\u003eHolotrichia serrata strains\u003c/em\u003e, the highest cellulolytic index was 1.49 ± 0.04 for the H4B3 isolate (\u003cem\u003eEnterobacter\u003c/em\u003e sp.), while the lowest index was 0.26 ± 0.012 for H4B2 (\u003cem\u003eEnterobacter ludwigii\u003c/em\u003e). The cellulolytic indices of the gut bacteria isolates are presented in Fig. 4A (Table S6). The cellulolytic indices varied significantly among the different bacterial strains, as indicated by the ANOVA results: F(2,12)=661.1, p\u0026lt;2×10−. Duncan’s test further confirmed these differences, revealing significant variation in the cellulolytic indices between the strains. Strain LC3B1 exhibited the highest cellulolytic index, followed by strain LC1B1. Based on these findings, four bacterial isolates—LC1B1, LC3B1, H4B3, and H5B2—were selected for further study due to their promising cellulolytic activities (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubstrate Degradation Ratio of Agricultural Residues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe four selected bacterial isolates were cultured in media containing powdered corncob, paddy straw, and groundnut husk substrates, followed by incubation for eight days. Among the isolates, H5B2 exhibited the highest degradation efficiency, reaching 48.15 ± 1.56% for groundnut husk, whereas LC3B1 exhibited 46 ± 0.608% for paddy straw powder and 43±0.27% for corncob. The substrate degradation ratio was significantly different between the F(2, 3)= 12.236 strain (p \u0026lt;0.05) and the F(2,2)=8.050 strain (p\u0026lt;0.05) but not between substrates. The other isolates also demonstrated significant degradation capabilities, as shown in Fig. 5 (Table S7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellulase (β-1,4-Endoglucanase) Enzyme Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCellulase (β-1,4-endoglucanase) activity assays were conducted using the DNS method at 50°C and pH 4. \u0026nbsp;β-1,4-endoglucanase was statistically significant among the various substrates F(2, 2)= 7.3, p \u0026lt;0.024 but not between strains of bacteria F(2,3)=1.16, p=0.39). Among the tested genes, LC3B1 had the highest beta 1,4-endoglucanase activity on ground nut husk, followed by LC1B1, which had the second highest endoglucanase activity (3.614 ± 0.02 U/mL), particularly on the corn cob powder substrate. LC1B1 also exhibited significant activity on groundnut husk (2.168 ± 0.07 U/mL), highlighting its potential for enzyme production. Isolates H4B3 (2.4 ± 0.03 U/mL) and H5B2 (1.626 ± 0.01 U/mL) also exhibited notable endoglucanase activities on groundnut husk. Additionally, LC1B1 exhibited activity (1.628 ± 0.01 U/mL) on paddy straw, whereas the remaining three bacteria displayed activities less than 0.7 U/mL on all three substrates. Moreover, LC3B1 exhibited considerable endoglucanase activity (2.082 ± 0.02 U/mL) on corncob.\u003c/p\u003e\n\u003cp\u003eThese findings underscore the substrate-specific cellulase activities of the bacterial isolates, with LC3B1 showing significant potential for enzyme production, particularly on groundnut husk and corncob substrates. The results are depicted in Fig. 6 (Table S8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR and SEM Results for the Breakdown of Cellulosic Bonds in Filter Paper\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural analysis of the four bacterial isolates was performed using attenuated total reflectance (ATR) mode in the infrared range of 400–4000 cm−1, enabling the detection of bond types and functional groups (Fig. 7). The peak at 1160 cm−1 is attributed to C-O-C stretching in the cellulose/hemicellulose molecules. The peak between 2896 and 2900 cm−1 indicates C-H (β-glycosidic bond) deformation in the polysaccharide molecules. The stretching of carbon and oxygen molecules between 1749 and 1599 cm−1 revealed decomposition products such as ketones, aldehydes, alcohols, and carboxylic acids derived from cellulose and hemicellulose. Additionally, the shifts in the O–H and C–H stretching vibrations at 3333 and 2894 cm−1 can be attributed to changes in the intra- and intermolecular bonds of the cellulosic polymer. The FTIR results revealed broad absorption bands at 897, 1651, and 2900 cm−1, corresponding to COC, CCO, OCH deformation, C-5, C-6 motion stretching, OH bending, and CH stretching, respectively. The intense peak at 3000–3700 cm−1 is attributed to the stretching of OH functional groups and amine groups.\u003c/p\u003e\n\u003cp\u003eField emission scanning electron microscopy (FESEM) provided insights into the significant structural changes in the surface morphology of both the control and treated filter paper (FP) samples (Fig. 8). Compared to those of the control substrates, the treated FP surface exhibited a rough appearance, likely due to bacterial adherence and superficial hydrolysis of the biomass. The bacterial cells adhered to the substrate, potentially creating pores and tunnels that allowed access to the internal fibers of the cellulosic biomass. This process suggested that the long cellulose chains in the FPs released their microfibers. The electron micrographs further demonstrated substrate disruption, as evidenced by the accumulation of hydrolyzed biomass on the surface.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInsects host a diverse array of microorganisms in their guts, which play vital roles in fulfilling the insects' physiological and ecological needs. These microorganisms enable insects to survive in harsh environmental conditions by aiding in digestion and the assimilation of food. Moreover, gut bacteria detoxify harmful substances and protect insects from pathogens by producing antimicrobial compounds (Jang and Kikuchi, 2020) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Lemke et al. (2003) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] conducted a comprehensive study on the microbiota of the white grub species \u003cem\u003ePachnoda ephippiata\u003c/em\u003e, finding that both the hindgut and midgut harbored diverse microorganisms involved in cellulose degradation, microbial fermentation, and proteolytic activities. Although numerous studies have reported the diversity and functional roles of bacteria in the guts of various white grub species [\u003cspan additionalcitationids=\"CR48 CR49 CR50 CR51\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], no reports exist on the bacterial diversity in \u003cem\u003eH. serrata\u003c/em\u003e and \u003cem\u003eL. coneophora\u003c/em\u003e, which are native to Karnataka, India.\u003c/p\u003e \u003cp\u003eIn this study, 17 bacterial isolates were obtained from the fermentation chambers of \u003cem\u003eL. coneophora\u003c/em\u003e and \u003cem\u003eH. serrata\u003c/em\u003e. Molecular characterization through 16S rRNA gene sequencing revealed significant diversity among the isolated bacterial strains, with the most dominant families being Enterobacteriaceae, followed by Bacillaceae and Pseudomonadaceae, aligning with previous findings in other white grub species [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Phylogenetic analysis further elucidated the evolutionary relationships among these isolates, revealing the presence of diverse taxa such as Firmicutes and Proteobacteria, consistent with other studies on white grub species [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe cellulolytic activity of these bacteria was assessed by measuring the zone of clearance surrounding bacterial colonies, indicative of carboxymethyl cellulose (CMC) hydrolysis by secreted cellulase (CMCase). Thirteen of the isolated bacteria exhibited significant cellulolytic activity. The isolate LC3B1 (\u003cem\u003eBacillus toyonensis\u003c/em\u003e) from \u003cem\u003eL. coneophora\u003c/em\u003e had the highest cellulolytic index (1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037), while H4B3 (\u003cem\u003eEnterobacter\u003c/em\u003e sp.) from \u003cem\u003eH. serrata\u003c/em\u003e showed the highest cellulolytic index (1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04). Additionally, substrate degradation abilities of selected bacterial strains demonstrated promising results, consistent with earlier reports on bacterial isolates from other white grub species [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Pretreatment of substrates before degradation was found to enhance degradation efficiency, with bacterial isolates from rumen showing higher efficiency than gut bacteria [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCellulase enzyme assays highlighted distinct substrate-specific activities among the bacterial isolates, emphasizing their potential for lignocellulosic biomass conversion [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. For instance, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eKlebsiella\u003c/em\u003e sp., and \u003cem\u003eBacillus\u003c/em\u003e sp. have been identified as significant cellulase producers in studies on bacterial cellulase from insect guts, with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e exhibiting endoglucanase activity at 3.5 U/mL [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and \u003cem\u003eKlebsiella variicola\u003c/em\u003e showing 0.092 U/mL cellulolytic activity [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In another study, cellulose-degrading bacteria from invertebrates such as bookworms, termites, snails, and caterpillars exhibited extracellular cellulase activities ranging from 0.012 to 0.196 IU/mL for filter paper cellulase (FPC) and from 0.162 to 0.400 IU/mL for endoglucanase assay [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The β-1,4-endoglucanase enzyme assay, as a preliminary test, is noteworthy for optimizing enzyme production under varying pH values, temperatures, substrates, and metal ion concentrations.\u003c/p\u003e \u003cp\u003eFurther insights into the breakdown of cellulosic bonds were obtained using FTIR and SEM, which confirmed the enzymatic destruction of cellulose by the isolated strains [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Functional group absorbance bands associated with structural changes in biomass were attributed to C-O, C\u0026thinsp;=\u0026thinsp;C, and C-C-O stretching at 1035 cm⁻\u0026sup1;, C-H stretching at 2840 cm⁻\u0026sup1; and 2937 cm⁻\u0026sup1;, O-H in-plane bending at 1440 cm⁻\u0026sup1;, and C\u0026thinsp;=\u0026thinsp;O stretching (unconjugated) at 1682 cm⁻\u0026sup1;, present in cellulose, hemicellulose, and lignin [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, this study is the first to identify cellulolytic bacteria from the gut microbiota of the white grub species \u003cem\u003eH. serrata\u003c/em\u003e and \u003cem\u003eL. coneophora\u003c/em\u003e. These bacteria exhibit significant potential for the degradation of complex, cellulose-rich organic wastes. The prevalence of cellulase-producing strains from families such as Enterobacteriaceae, Bacillaceae, and Pseudomonadaceae highlights their crucial role in lignocellulosic biomass degradation. The variability in cellulose degradation efficiency across different bacterial strains and substrates underscores the substrate-specific nature of cellulose breakdown.\u003c/p\u003e \u003cp\u003eThe cellulose degradation process yields reducing sugars like glucose, which are essential for bioethanol production. Utilizing these reducing sugars can reduce dependence on traditional raw materials like sugar and corn, which are commonly used in conventional ethanol production. Effective lignocellulose degradation thus requires the application of efficient microbial strains. Consequently, these findings underscore the potential for sustainable waste management and biofuel production through bioconversion techniques that leverage cellulolytic bacteria isolated from white grub guts. This approach could significantly enhance the sustainability and value of biofuel production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRBN acknowledges the Science and Engineering Research Board, New Delhi for the Fund TAR/2020/000014. RBN acknowledges ICAR-CIPHET and Ludhiana for providing support. RBN thanks T. Govindaraju of JNCASR for supporting the FTIR and SEM studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u0026nbsp;Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; The insects were predominantly pestiferous and were collected from farmer fields after providing consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u0026nbsp;Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u0026nbsp;Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe data were submitted to NCBI under accession numbers\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"632\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTRAIN ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003e\u003cstrong\u003eACCESSION\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNUMBER\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSPECIES\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCLOSEST STRAIN IN GENBANK\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDENTITY (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eLeucophalis coneophora\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC1B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella\u003c/em\u003e sp. HaMC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e99.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC1B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus siralis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus siralis\u0026nbsp;\u003c/em\u003eJ28TS8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e99.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC2B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542505\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus toyonensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus toyonensis\u0026nbsp;\u003c/em\u003estrain MB14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC2B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus cereus\u0026nbsp;\u003c/em\u003estrain ABC10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC2B3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella oxytoca\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella oxytoca\u0026nbsp;\u003c/em\u003estrain S1-2-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e98.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC3B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter farmeri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter farmeri\u003c/em\u003eisolate CCB19B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e97.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\" valign=\"top\"\u003e\n \u003cp\u003eCDBLC6B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\" valign=\"top\"\u003e\n \u003cp\u003ePP542500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pasteurii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pasteurii\u0026nbsp;\u003c/em\u003estrain SPARK1448C2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e98.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eHolotrichia serrata\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH2B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e sp.strain H374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e99.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH3B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas monteilii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas monteilii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e99.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH3B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas urethralis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas urethralis\u0026nbsp;\u003c/em\u003estrain BML-PP042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e99.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH3B3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542518\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eAchromobacter piechaudii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eAchromobacter piechaudii\u0026nbsp;\u003c/em\u003estrain ERG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e93.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH3B4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter koseri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter\u003c/em\u003e\u003cem\u003e\u0026nbsp;koseri\u0026nbsp;\u003c/em\u003estrain IHB B 6842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e98.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH4B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542525\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter tabaci\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter tabaci\u0026nbsp;\u003c/em\u003estrain TBMAX92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e96.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH4B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter ludwigii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter ludwigii\u0026nbsp;\u003c/em\u003estrain AA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e96.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH4B3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter\u003c/em\u003e sp.JBIWA003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e97.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH5B1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542527\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter\u003c/em\u003e sp.strain LFR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.873417721518987%\"\u003e\n \u003cp\u003eCDBH5B2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.088607594936708%\"\u003e\n \u003cp\u003ePP542526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.943037974683545%\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.90506329113924%\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter\u003c/em\u003e\u003cem\u003e\u0026nbsp;baumannii\u0026nbsp;\u003c/em\u003estrain JC359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.189873417721518%\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u0026nbsp;Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;E. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Science and Engineering Research Board under Teacher Associateship Research Excellence, TAR/2020/000014\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u0026nbsp;Authors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRBN and PKV: conceptualization of work\u003c/p\u003e\n\u003cp\u003eGVV- Conducted the experiment and collected the data\u003c/p\u003e\n\u003cp\u003ePKV- and GVV-Collected insects for the study\u003c/p\u003e\n\u003cp\u003ePKV-Identification of white grubs and dissection of the fermentation chamber\u003c/p\u003e\n\u003cp\u003eRBN- Supervised overall study and edited the manuscript\u003c/p\u003e\n\u003cp\u003eBP- Corrected the manuscript\u003c/p\u003e\n\u003cp\u003eGKR- Involved in collecting the experimental data\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSteinhaus EA. 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Characterization of cellulosic fibers by FTIR spectroscopy for their further implementation to building materials. AJAC. 2018;9(6), 303-310\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-4958316/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4958316/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Abstract\nThe gut microbiota of insects plays a crucial role in digesting food, providing nutrients, and synthesizing enzymes. This approach is particularly relevant for degrading lignocellulosic biomass and managing waste. In Karnataka, the larvae of Holotrichia serrata and Leucopholis canephora are major crop pests, but the role of their bacterial communities in lignocellulose degradation has not been well studied. This study aimed to isolate and evaluate bacteria from these larvae for their ability to degrade lignocellulose.Approximately seventeen cellulolytic bacterial strains were isolated from the fermentation chamber of white grubs, primarily from the Firmicutes and γ-proteobacteria classes. Notable species included Bacillus, Enterobacter, and Klebsiella. Bacillus toyonensis strain LC3B1 demonstrated significant cellulolytic activity, with a cellulolytic index of 1.93 ± 0.037. The degradation of corncob powder was the highest (28.15 ± 1.56%), followed by that of paddy straw powder (31.45 ± 0.608%) and groundnut husk powder (33.25 ± 0.823%), indicating the strong ability of these powders to degrade agricultural residues. FTIR analysis of the substrate carboxymethyl cellulose (CMC) hydrolyzed by LC3B1 revealed decomposition products such as ketones, aldehydes, alcohols, and carboxylic acids. Scanning electron microscopy (SEM) revealed significant morphological changes and the formation of pores and tunnels in the treated biomass.The diverse cellulolytic capabilities of gut bacteria from white grubs, including those of the Bacillaceae, Enterobacteriaceae, and Pseudomonadaceae families, offer promising opportunities for lignocellulosic biomass degradation, biofuel production, and sustainable waste management.","manuscriptTitle":"Evaluation of Cellulolytic Gut Bacteria Isolated from White Grubs (Holotrichia serrata and Leucopholis coneophora) and Their Utilization in Lignocellulose Degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-23 09:21:00","doi":"10.21203/rs.3.rs-4958316/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":"288e32dd-4b18-436a-a432-b0cf6a928a09","owner":[],"postedDate":"September 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T09:21:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-23 09:21:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4958316","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4958316","identity":"rs-4958316","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}