Evaluation of different culture media for growth and sporulation of Bacillus thuringiensis PDKV-Bt-SY-4 under submerged and static fermentation conditions

Introduction

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming entomopathogenic bacterium that is widely used as a microbial insecticide for the management of economically important insect pests (Bravo et al., 2007; Palma et al., 2014) [1,2] . During sporulation, Bt produces parasporal crystalline proteins, mainly Cry and Cyt toxins, which show insecticidal activity against susceptible insects belonging to several orders, particularly Lepidoptera, Coleoptera and Diptera (Bravo et al., 2007; Palma et al., 2014) [1,2] . Following ingestion by a susceptible insect, these toxins are activated under the alkaline conditions of the midgut and bind to specific receptors on the midgut epithelial cells. This interaction results in disruption of the cell membrane, cellular damage and, ultimately, death of the insect (Bravo et al., 2007; Pardo-López et al., 2013) [1,3] . The specificity of Bt toxins, together with their comparatively favourable environmental profile, has contributed to the widespread use of Bt in biological and integrated pest management programmes (Palma et al., 2014) [2] . The effectiveness of a Bt-based biopesticide is influenced by several factors, including the bacterial strain, composition of the culture medium, fermentation conditions, level of spore production and formulation characteristics (Duraisamy et al., 2023; Khedher et al., 2011) [4,5] . Optimizing these factors is therefore important for obtaining preparations with a high viable spore concentration, effective insecticidal activity and adequate storage stability (Duraisamy et al., 2023; Khedher et al., 2011) [4,5] . Among these factors, the selection of a suitable culture medium and fermentation condition is particularly important because they directly influence bacterial growth and sporulation (Duraisamy et al., 2023; Khedher et al., 2011) [4,5] . Therefore, the present study was undertaken to evaluate different culture media for growth and sporulation of B. thuringiensis PDKV-Bt-SY-4 under submerged and static fermentation conditions, with the aim of identifying a suitable production condition for subsequent development of a Bt-based biopesticide formulation.

Materials and Methods

The study was conducted to evaluate different media for the growth and sporulation of Bacillus thuringiensis PDKV-Bt-SY-4 under submerged and static fermentation conditions. The strain isolated and cultured from Department of Entomology and maintained at Laboratory of Molecular Biology and Nanotechnology, were used in the study. The study was conducted in Laboratory of Molecular Biology and Nanotechnology, Biotechnology Centre, Department of Agril. Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola (M.S.) during the year 2025-2026.

Culturing of Bt strains

The local Bacillus thuringiensis isolate PDKV-Bt-SY-4, previously isolated and screened for insecticidal activity in the Biotechnology Centre, was used in the present study. The isolate was subculture and maintained on Luria broth medium. Luria broth medium, as described by Travers et al. (1987) [6] , was used for the routine cultivation and maintenance of the B. thuringiensis isolate throughout the study.

Culture media used for fermentation

Different culture media were evaluated to determine their suitability for the growth and sporulation of Bacillus thuringiensis PDKV-Bt-SY-4 under static and submerged fermentation conditions. The media evaluated included Nutrient Carbon Mineral (NCM) medium, T3 medium, NCM supplemented with tyrosine and CuSO₄, Nutrient Yeast Salt Medium (NYSM), Glucose-based medium (GBM), and Tryptic Soy Broth (TSB). The respective media were prepared according to the formulations reported by Talkhan et al. (2013) [7] , Travers et al. (1987) [6] , Bora et al. (2024) [8] , Khedher et al. (2011) [5] , and Duraisamy et al. (2023) [4] as shown in Table 1.

Culture maintenance

The local isolate PDKV-Bt-SY-4, previously isolated and screened for insecticidal activity in the Biotechnology Centre, Department of Agril. Botany and Department of Entomology, was used for the present study. The culture was subculture and maintained on Luria Broth (LB) medium at 4 °C under aseptic conditions. The maintained culture was periodically subculture at every 15 days to ensure viability and purity before use in subsequent experiments. Freshly grown cultures were used for seed culture preparation and fermentation experiments.

Seed culture preparation of Bacillus thuringiensis PDKV-Bt-SY-4

The maintained culture of PDKV-Bt-SY-4 was used for the preparation of seed culture. A loopful of the actively growing culture was aseptically transferred into 50 mL of sterile Luria Broth (LB) medium in a 250 mL Erlenmeyer flask. The inoculated medium was incubated at 28 °C for 12 h or overight under shaking conditions at 180 RPM to obtain an actively growing culture with OD value more than 0.6. The resulting seed culture was used as the inoculum for subsequent fermentation experiments at a 5% (v/v) inoculum level.

Inoculation of seed culture into different fermentation media

The prepared seed culture of PDKV-Bt-SY-4 was used to inoculate the selected fermentation media. 50 mL of each medium were dispensed into sterile 250 mL Erlenmeyer flasks and inoculated aseptically with 5% (v/v) seed culture. An uninoculated flask containing the respective medium was maintained as a control. Each treatment was maintained in replication to ensure the reliability and reproducibility of the observations. The inoculated flasks were incubated at 28 °C for 5 days under two fermentation conditions. For submerged fermentation, the cultures were incubated on a rotary shaker at 180 rpm, whereas for static fermentation, the cultures were incubated without agitation. The cultures were subsequently evaluated for total viable cell count (TVCC) and viable spore count to compare the growth and sporulation of PDKV-Bt-SY-4 among the different media and fermentation conditions.

The viable bacterial population of Bacillus thuringiensis PDKV-Bt-SY-4 was estimated by the serial dilution and plate count method. A 0.9% sterile saline solution was used as the diluent for preparing serial ten-fold dilutions of the fermentation broth. An appropriate dilution was selected, and a measured aliquot was aseptically spread onto suitable agar medium. The inoculated plates were incubated under suitable conditions, and colonies developed on plates containing a countable number of colonies were enumerated. The viable bacterial population was expressed as colony-forming units per millilitre (CFU/mL) and calculated using the dilution factor and volume of sample plated (Cappuccino and Welsh, 2017).

Calculation

CFU/ml = ( Number of colonies × Dilution factor ) Volume of culture plate (in ml)

Estimation of total viable cell count (TVCC) and viable spore count

The total viable cell count (TVCC) and viable spore count of PDKV-Bt-SY-4 were determined by the serial dilution and plate count method. Sterile 0.9% saline solution was used as the diluent for preparing serial ten-fold dilutions of the fermentation broth.

For TVCC estimation, no heat treatment was applied. Appropriate serial dilutions of the culture were directly plated onto suitable agar medium. Following incubation, the colonies developed on countable plates were enumerated and expressed as CFU/mL, representing the total viable population, including vegetative cells and heat-resistant spores.

For viable spore enumeration, a separate aliquot of the fermentation broth was subjected to heat treatment at 80 °C for 10 min to eliminate the vegetative cells while retaining viable heat-resistant spores. The heat-treated sample was subsequently serially diluted using sterile 0.9% saline solution and plated onto suitable agar medium. After incubation, the colonies that developed were counted and expressed as CFU/mL (viable spores). The use of an 80 °C heat treatment for 15 min before serial dilution and plating has been reported for the selective enumeration of viable Bt spores.

Results and Discussion

Total viable cell count under submerged and static fermentation

The total viable cell count (TVCC) of Bacillus thuringiensis PDKV-Bt-SY-4 varied considerably among the different culture media and fermentation conditions. The results are presented in Table 2.

Under submerged fermentation (180 rpm), the highest TVCC was recorded in NYSM medium (3.2 × 10⁸ CFU/mL), followed by T3 medium (9.5 × 10⁷ CFU/mL), GBM (7.7 × 10⁷ CFU/mL), NCM medium (7.3 × 10⁷ CFU/mL), NCM+ medium (7.2 × 10⁷ CFU/mL), and TSB (1.9 × 10⁷ CFU/mL). Thus, NYSM supported the highest bacterial growth under submerged fermentation. Under static fermentation, the highest TVCC was observed in NCM medium (1.12 × 10⁸ CFU/mL), followed by NCM+ (7.6 × 10⁷ CFU/mL), NYSM (6.6 × 10⁷ CFU/mL), T3 (4.9 × 10⁷ CFU/mL), TSB (4.1 × 10⁷ CFU/mL), and GBM (3.6 × 10⁷ CFU/mL). Overall, submerged fermentation resulted in higher TVCC than static fermentation in four of the six media, namely T3, NYSM, GBM, and TSB. The most pronounced difference was observed in NYSM, where the TVCC under submerged fermentation (3.2 × 10⁸ CFU/mL) was substantially higher than that under static fermentation (6.6 × 10⁷ CFU/mL). In contrast, NCM showed better bacterial growth under static conditions (1.12 × 10⁸ CFU/mL) than under submerged conditions (7.

Viable spore count under submerged and static fermentation

The viable spore count of Bacillus thuringiensis PDKV-Bt-SY-4 varied among the different culture media and fermentation conditions. The results are presented in Table 3. Under submerged fermentation (180 rpm), the highest viable spore count was recorded in NYSM (1.5 × 10⁸ CFU/mL), followed by NCM (1.14 × 10⁸ CFU/mL), NCM+ (5.4 × 10⁷ CFU/mL), GBM (3.7 × 10⁷ CFU/mL), T3 (3.6 × 10⁷ CFU/mL) and TSB (3.5 × 10⁷ CFU/mL). Thus, NYSM supported the highest spore production under submerged fermentation. Under static fermentation, the highest viable spore count was observed in NCM+ (1.32 × 10⁸ CFU/mL), followed by NCM (9.6 × 10⁷ CFU/mL), T3 (3.3 × 10⁷ CFU/mL), NYSM (3.1 × 10⁷ CFU/mL), GBM (2.4 × 10⁷ CFU/mL) and TSB (2.2 × 10⁷ CFU/mL). Therefore, NCM+ exhibited the highest spore production under static conditions. A comparison of the two fermentation conditions showed that NYSM and NCM supported higher viable spore production under submerged fermentation, whereas NCM+ showed considerably higher spore production under static fermentation. The difference was particularly evident in NYSM, where the viable spore count under submerged fermentation (1.5 × 10⁸ CFU/mL) was much higher than that observed under static fermentation (3.1 × 10⁷ CFU/mL). In contrast, NCM+ recorded a higher viable spore count under static fermentation (1.32 × 10⁸ CFU/mL) than under submerged fermentation (5.4 × 10⁷ CFU/mL). Among all the treatments evaluated, the highest overall viable spore count was recorded in NYSM under submerged fermentation (1.5 × 10⁸ CFU/mL), while the lowest count was observed in TSB under static fermentation (2.2 × 10⁷ CFU/mL). The results indicate that spore production was influenced by both the culture medium and the fermentation condition. NYSM was more favourable for spore production under submerged conditions, whereas NCM+ performed better under static conditions.(

Conclusion

The comparative evaluation of different culture media under submerged and static fermentation conditions was carried out to identify a suitable medium and fermentation condition for the efficient production of Bacillus thuringiensis PDKV-Bt-SY-4. The cultures were incubated for 5 days (120 h) at 28 °C, with submerged fermentation carried out at 180 rpm, while static fermentation was performed without agitation (0 rpm). The performance of the different media was evaluated based on total viable cell count (TVCC) and viable spore count.

Graph 1. Evaluation of Total viable cell counts of Bacillus thuringiensis PDKV-Bt-SY-4 in different culture media under submerged and static fermentation.

Under submerged fermentation, NYSM recorded the highest TVCC (3.2 × 10 8 CFU/mL) and viable spore count (1.5 × 10 8 CFU/mL). In comparison, under static fermentation, NCM recorded the highest TVCC (1.12 × 10 8 CFU/mL), while NCM supplemented with L-tyrosine and CuSO₄ (NCM+) recorded the highest viable spore count (1.32 × 10 8 CFU/mL). Although NYSM showed the highest overall growth and spore production under submerged conditions, the difference between submerged and static fermentation was not consistently large across all media. In particular, NCM and NCM+ showed relatively comparable performance between the two fermentation conditions. From a practical and economic perspective, static fermentation has an important advantage because it does not require continuous agitation, thereby reducing energy consumption, equipment requirements, and overall production costs. Since the objective of the present study was to develop a cost-effective and farmer-friendly B. thuringiensis production system, avoiding mechanical agitation wherever possible would be beneficial. Therefore, when the difference in growth and sporulation between submerged and static fermentation is relatively small, static fermentation can be preferred over submerged fermentation for further development. Based on the results obtained under static fermentation, NCM+ showed the highest viable spore count (1.32 × 10 8 CFU/mL), while NCM showed the highest TVCC (1.12 × 10 8 CFU/mL). Since spore production is particularly important for the development of a stable B. thuringiensis biopesticide, NCM+ under static fermentation was considered the most promising condition for further studies, subject to confirmation through statistical analysis and subsequent experiments. Thus, considering biological performance together with the economic advantage of eliminating agitation, static fermentation was considered a suitable approach for the subsequent development of the B. thuringiensis PDKV-Bt-SY-4 formulation. The selected static fermentation condition can potentially simplify the production process and reduce production costs while maintaining satisfactory bacterial growth and spore production.

Acknowledgement

AcknowledgementsThe authors are thankful to the Head, Biotechnology Centre, Department of Agril. Botany, Dr. PDKV, Akola, Maharashtra, India and the Associate Dean, Post Graduate Institute, Dr. PDKV, Akola (MS) for providing all the basic facilities required for conduction of the experiment. Graph 2. Evaluation of viable spore counts of Bacillus thuringiensisPDKV-Bt-SY-4 in different culture media under submerged and static fermentation

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