Isolation and identification of cellulolytic bacteria from the gut of white grub native to the Kolhapur region

Introduction

Cellulose is a natural carbohydrate (a polysaccharide) made of glucose units linked together. It is the main structural polysaccharide of plants and predominant constituent of cell wall of plant cells. Its content ranges approximately 35 to 50 per cent of plant dry weight. This polysaccharide is composed of β-D-glucose units joined by glycosidic linkage. Cellulose is commonly degraded by an enzyme called cellulase (Singh et al., 2023) [13] . Cellulolytic bacteria are capable of producing cellulase enzymes that catalyze the hydrolysis of cellulose into simpler sugars. In recent years, cellulolytic bacteria have emerged as important biological resources for sustainable management of agricultural residues and environmental friendly biomass conversion technologies (Gupta et al., 2011) [6] . The insect gut microbiota performs several important functions including cellulose degradation, nutrient acquisition, detoxification of plant metabolites and maintenance of host fitness (Engel and Moran, 2013) [5] . Due to continuous adaptation to cellulose-rich diets, gut microorganisms frequently possess highly efficient enzymatic systems for lignocellulose degradation. Recent studies have demonstrated that insect digestive systems harbour diverse cellulolytic bacterial groups exhibiting significant cellulase activity (Dar et al., 2021; Schroeder et al., 2022) [4,11] . Therefore, insect gut microbiota has become an important target for the isolation of novel cellulolytic bacteria with agricultural and industrial significance. Among the various insect groups investigated, white grubs have attracted considerable attention because of their feeding behaviour and digestive capabilities. White grubs are the larval stages of scarab beetles belonging to the family Scarabaeidae and are widely distributed in agricultural ecosystems. Species such as Holotrichia serrata Fabricius and Leucopholis lepidophora Blanchard are common in Maharashtra and are considered important pests of sugarcane and groundnut. These grubs spend most of their life cycle beneath the soil surface and feed continuously on roots, decaying organic matter and cellulose-rich plant materials (Bhavani et al., 2024) [1] . The remarkable ability of white grubs to survive on celluloserich diets suggests the presence of efficient cellulolytic microorganisms within their digestive systems. Previous studies have reported that a substantial proportion of ingested cellulose undergoes degradation within the gut of scarab larvae due to microbial activity (Shengwei et al., 2012) [12] . Despite the growing interest in insect-associated cellulolytic microorganisms, information on cellulolytic bacteria associated with the gut of white grubs native to the Kolhapur region of Maharashtra is limited. Isolation and preliminary screening of such bacteria can help identify promising cellulolytic isolates, while molecular characterization provides reliable identification at the bacterial taxonomic level. Therefore the present study provides preliminary information on cellulolytic bacteria associated with white grub gut and establishes a basis for their further characterization and evaluation.

Materials and Methods

Insect Sample Collection

The third instar larvae of white grub species were collected from infested fields in Vasagade (16.67 o N latitude and 74.34 o E longitude) and Haldi (16.58 o N and 74.16 o E longitude) village in Kolhapur region. These white grubs larvae were subsequently transported to the Entomology laboratory of R. C. S. M. Govt. College of Agriculture, Kolhapur in sterilized aerated plastic containers. Nearly 60 white grubs larvae of two white grub species were collected and dissected for experiment.

Insect Dissection

The collected grubs were rinsed by distilled water for 30 seconds followed by 70 per cent ethanol for 60 seconds. Again it was rinsed by distilled water for 30 seconds to remove the disinfectant (Bhavani et al., 2024) [1] . The sterilized white grub was mounted on wax dish. The sterilized grubs were dissected under laminar airflow cabinet using sterile microscissors and forceps to extract the gut, specifically isolating and removing the fermentation chamber to extract the gut portion. Different gut sections were incised and separated. The dissected gut section was transferred to sterilized 1.5ml microcentrifuge tubes containing 0.85 per cent NaCl and macerated and used for serial dilution.

Isolation of Cellulose Decomposing Bacteria

The cellulolytic media were used for isolation of cellulose decomposing bacteria. Isolation of pure culture of cellulose decomposing bacteria was carried out from dissected gut section on cellulolytic medium. Serial dilution were made from 10 -1 to 10 -7 dilution. Subsequently, the autoclaved and cooled cellulolytic medium was poured in sterile glass petri plates (approx.. 20ml/plate) and were allowed to solidify. The serially diluted content from 10 -1 to 10 -7 was plated with the help of sterilized glass loop in petri plates containing celluloytic media. The bacterial plates were incubated for 24-48 hrs. at 37 ℃ and 65±5 per cent RH (Bhavani et al., 2024) [1] . Bacterial colonies were selected based on the difference in colour, size and morphology. A single representative isolate of each morphotype was selected and purified by streaking on cellulolytic medium plates repeatedly until the pure bacterial culture was obtained. The purified bacterial colonies were subcultured on agar slants.

Screening of Cellulolytic Activities of Bacterial Isolates

The ability of the bacterium to degrade cellulose were tested on a CMC (Carboxymethyl cellulose) agar medium with the following composition: The bacterial colonies were transferred on CMC agar medium to check its activity. The bacterial plates were incubated at 28 ℃ for 4 days and then treated with Congo red i.e. bacterial plates prepared with CMC agar medium were flooded with an aqueous solution of 0.2 per cent Congo red (w / v) for 30 min to confirm the presence or absence of endoglucanase activity. After 30 min, the Congo red solution was poured off and plates were treated by flooding with 1 M NaCl for 15 min. The NaCl solution then poured off and the degradation of CMC was observed as a clear halo around the bacterial colony (Danu et al., 2022) [3] . The cellulolytic index was calculated by the following formula (Bhavani et al., 2024) [1] .

Cellulolytic index =

Diameter of zone of clearance -Diameter of bacterial colony

Diameter of bacterial colony

Among the purified bacterial isolates, the isolate exhibiting the highest cellulolytic index on CMC agar medium was selected for molecular identification.

Genomic DNA Extraction

A pure culture of isolate ACK was grown in appropriate bacterial growth medium. Bacterial cells were harvested and genomic DNA was extracted using the DNA extraction procedure followed in the laboratory of Agharkar Research Institute. The extracted genomic DNA was assessed for quality and suitability for PCR amplification.

Amplification of 16S rRNA gene

The bacterial 16S rRNA gene was amplified using universal bacterial primers. Genomic DNA obtained from isolate ACK was used as the template for PCR amplification. The PCR product was examined by agarose gel electrophoresis. The amplified product was subsequently purified and submitted for sequencing.

16S rRNA gene sequencing

The purified PCR product of isolate ACK was subjected to nucleotide sequencing. The resulting sequence was processed and used for molecular identification. A partial 16S rRNA gene sequence of 445 bp was obtained from isolate ACK.

Molecular Identification

The obtained nucleotide sequence was compared with sequences available in the NCBI GenBank database using BLASTn. Based on sequence similarity and taxonomic comparison, isolate ACK was assigned to the genus Hydrogenophaga and designated: Hydrogenophaga sp. strain ACK

Sequence Submission to GenBank

The partial 16S rRNA gene sequence of isolate ACK was submitted to the NCBI GenBank database. The sequence was assigned accession number: PZ783938

The GenBank record describes it as Hydrogenophaga sp. strain ACK 16S ribosomal RNA gene, partial sequence, with a sequence length of 445 bp.

Result and discussion

Isolation of Cellulolytic Bacteria from the Gut of White Grubs

The gut of white grubs harbours a diverse community of microorganisms that plays an important role in the degradation of cellulosic plant materials. Among these microorganisms, cellulolytic bacteria produce cellulase enzymes that hydrolyzed cellulose into simple sugars, thereby contributing to digestion of plant biomass. cellulolytic bacteria were isolated from the fermentation chamber of the hindgut of white grub (Holotrichia serrata) collected from the Kolhapur region of Maharashtra. The isolation of cellulolytic bacteria was done on cellulolytic medium by serial dilution and pour plate technique. Carboxymethyl Cellulose (CMC) served as the sole carbon source in the medium, thereby selectively supporting the growth of cellulolytic bacteria capable of producing cellulase enzymes while suppressing noncellulolytic microorganisms. Therefore, the bacterial isolates recovered on CMC medium were considered potential cellulolytic bacteria. Three distinct cellulolytic bacterial isolates were successfully obtained from the 10 -5, 10 -6 and 10 -7 serial dilution plates. The isolates were named as HS-1, HS-2 and LL-1. These findings are in close agreement with Bhavani et al.

(2024) [1] , who isolated 17 cellulolytic bacterial isolates from the fermentation chamber of white grub. Among these 7 isolates were derived from Leucopholis coneophora, while 10 were obtained from Holotrichia serrata. Similarly, Shengwei et al. (2012) [12] isolated 207 strains of aerobic and facultatively anaerobic cellulolytic bacteria from the gut of Holotrichia parallela larvae. Handique et al. (2017) [7] found 30 cellulolytic bacterial isolates from the gut of Lepidiota mansueta larvae.

Screening of Cellulolytic Bacteria

The bacterial isolates obtained from the gut of white grubs were screened for cellulolytic activity on Carboxymethyl Cellulose (CMC) agar medium using the Congo red staining method. Cellulase producing bacteria hydrolyzed the cellulose present in the medium, resulting in the formation of clear halos around the bacterial colonies after staining. The appearance of these hydrolysis zones confirmed the production of extracellular cellulase enzymes by the isolates.

The cellulolytic index was calculated by observing diameter of zone of clearance and diameter of bacterial colony. Among the 3 bacterial isolates examined, 2 demonstrated cellulolytic activity. The maximum cellulolytic index of 2.1 was exhibited by isolate HS-1. The isolate HS-2 shows 1.4 cellulolytic index. The isolate LL-1 did not shows any clear halo around bacterial colony. Based on these results, isolate HS-1 was chosen for further molecular identification because of their promising cellulolytic activities. The cellulolytic indices of the gut bacteria isolated are represented in the Table 3 The present findings are in accordance with Gupta et al.

(2011) [6] who found 8 bacterial isolates positive on screening media. Similarly, Handique et al. (2017) [7] tested 30 bacterial strains for cellulolytic activity using CMC agar medium. Of these strains, 5 degrade cellulose. Biswas et al.

(2019) [2] screened 19 cellulolytic bacterial isolates from the gut of two insect species on CMC agar medium. [1,8,9] screened the bacterial isolates for cellulolytic activity using Congo red staining on CMC agar medium.

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