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

doi:10.21203/rs.3.rs-4958316/v1

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

https://doi.org/10.21203/rs.3.rs-4958316/v1

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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.

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].

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.

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.

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.

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 farmeriisolate 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

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

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Abstract

Figures

Background

Material and methods

Substrate Preparation

Insect sample collection

Isolation of Gut Bacteria

Molecular Characterization and Identification of Isolated Bacterial Strains:

Screening of cellulose-degrading bacteria

Substrate Degradation Ratio

Cellulase (β-1,4-Endoglucanase) Enzyme Assay

SEM Analysis of the Hydrolyzed Substrate

FTIR Analysis of the Hydrolyzed Substrate

Statistical Analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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