Characterization of the Bacterial Development and Antifungal Properties of Bacillus thuringiensis var. kurstaki HD-1 Obtained by Bioconversion of Agroindustrial Effluents

Abstract

Large-scale production of Bacillus thuringiensis, one of the most widely used biopesticides, is often limited by the high cost of conventional culture media. In this study, fermented cassava paste water (EFM), ripe mango pulp juice (CM), and cashew apple juice (JPC) were evaluated as alternative substrates for the liquid fermentation of B. thuringiensis var. kurstaki HD-1. Physicochemical analyses revealed acidic pH values and classified the substrates into two clusters: CM with high C/N ratios, organic matter, total sugars, and proteins, and EFM and JPC with lower C/N ratios and nutrient levels. Fermentation results indicated that JPC supported the highest biomass production (8.29 × 10¹³ CFU mL⁻¹), exceeding that in the standard Tryptone Soy Broth (TSB) medium. However, CM promoted the highest sporulation rate (1.46 × 10¹³ CFU mL⁻¹) and the greatest bioactive lipopeptides—iturins (102.2 mg L⁻¹) and surfactins (554.7 mg L⁻¹)—surpassing TSB. The antifungal activity of crude fermented CM, EFM, and TSB was evaluated against Sclerotium rolfsii. All samples significantly inhibited mycelial growth of the pathogen with no significant differences among substrates or concentrations tested. This study highlights the potential of B. thuringiensis-fermented agrowaste as a cost-effective, environmentally friendly biocontrol tool for Sclerotium rolfsii.

1. Introduction

Since the Green Revolution of the 1960s, synthetic chemical pesticides have played a central role in protecting crops from pests and pathogens, significantly reducing yield losses and supporting the growth of agricultural economies worldwide, including Côte d’Ivoire’s. For proof, tomato is an important vegetable crop in Côte d’Ivoire, both for household consumption and income generation. According to FAO data, national tomato production reached approximately 60,300 tons in 2024, reflecting its growing role in the horticultural sector [1]. Many factors can be cited to explain this low production, notably biotic factors. Indeed, tomatoes are susceptible to different fungi, bacteria, and viruses. Among the most widespread parasitic attacks on tomatoes in Côte d’Ivoire, sclerotinia rot, caused by Sclerotium rolfsii (S. rolfsii), is even more common, both for its frequency and the extent of its host range. To reduce losses caused by this pathogen, many control methods, including chemical control, have been explored for a long time.

However, over the past few decades, mounting evidence has shown that intensive use of synthetic pesticides has led to severe adverse effects on ecosystems, human health, and long-term agricultural sustainability. Excessive pesticide inputs contaminate soil, water, and air [2], while also disrupting key ecosystem services such as biological pest regulation through the decline of pollinators and natural enemies [2,3].

From a public health perspective, the World Health Organization (WHO) estimates that approximately 385 million cases of unintentional acute pesticide poisoning occur annually, mainly in developing countries [4]. Chronic exposure to these compounds has been associated with endocrine disruption, cancer, and neurological disorders [5]. Moreover, repeated pesticide use promotes the emergence of resistant pest populations, reducing pesticide efficacy and creating a feedback loop of increased application rates and chemical diversification [6]. This dependency poses not only environmental and health risks but also economic challenges for smallholder farmers, particularly in low-income regions [7,8,9,10].

Given these drawbacks, integrated pest management (IPM) strategies are increasingly emphasizing the use of eco-compatible alternatives, particularly biopesticides, to reduce reliance on synthetic chemicals. Among available biological control agents, Bacillus thuringiensis (B. thuringiensis) is one of the most widely used due to its broad-spectrum insecticidal activity, environmental safety, and biodegradability [11,12,13]. Bt-based products account for more than 70% of the global bioinsecticide market [11,12], and Bt crops have substantially contributed to global yield improvement [13]. In Côte d’Ivoire, preliminary trials against Sahlbergella singularis demonstrated the effectiveness of B. thuringiensis var. kurstaki HD-1 (Btk HD-1) under field conditions [14].

Despite these advantages, the industrial production of B. thuringiensis remains limited by the high cost of conventional culture media [15]. In other parts, large quantities of agro-industrial waste are abandoned in the environment, incinerated, or disposed of in uncontrolled landfills, thereby contributing to soil, water, and air pollution. The FAO reported 250 million tons of inedible plant waste from various crop treatments in 2013 as agro-industrial waste [16]. The production of agro-industrial waste from various sources in India exceeds 350 million tons per year [17]. In addition, biomass, also known as biowaste, accounts for about 32% in high-income countries and 53–56% in middle- and low-income countries [18]. A significant portion of global waste is generated by agro-industrial residues from the industrial processing of agricultural by-products [19]. This waste, often neglected or poorly managed, is nevertheless a valuable resource in the transition towards a circular and sustainable economy. Faced with the environmental, economic, and social issues related to organic waste management, the valorization of agricultural and agro-industrial waste is a priority, particularly in countries with a strong agro-export vocation such as Côte d’Ivoire. The abundance of waste, its profitable availability, and management problems have led to ongoing research into fermentation methods [20]. In recent years, biotechnological innovations have highlighted the potential of converting agricultural, industrial, or domestic residues into valuable bioproducts. Agro-industrial effluents and municipal sludge, often considered waste management challenges, can be transformed into nutrient-rich substrates suitable for microbial fermentation. Several non-conventional media—such as rice bran, banana pseudostem residues, starch-processing wastewater, and sewage sludge—have already been evaluated for B. thuringiensis production, with promising results [16,17,18,21,22].

Building on these advances, the present work aims to study the bacterial development and antifungal properties of B. thuringiensis var. kurstaki HD-1, obtained by bioconversion of three agro-industrial liquid wastes—fermented cassava paste water, ripe mango pulp, and cashew apple juice—as alternative substrates. The overarching objective is to develop an efficient and sustainable bioprocess for producing a B. thuringiensis-based biopesticide, thereby contributing to both environmental protection and the principles of a circular bioeconomy.

2. Materials and Methods

2.1. Collection of Agro-Industrial Effluents

Three agro-industrial liquid residues were evaluated as potential low-cost culture media to produce B. thuringiensis var. kurstaki HD-1 (Btk HD-1). The selected substrates were

(i).

Fermented cassava paste water (EFM);

(ii).

Ripe mango pulp (CM);

(iii).

Cashew apple juice (JPC).

These effluents were collected from small-scale processing units located in Yamoussoukro, central Côte d’Ivoire. Selection was based on their expected nutrient composition and mineral content, both of which are critical for supporting B. thuringiensis growth and sporulation [19,20,23]. The collected samples were stored in sterilized polypropylene containers and transported to the laboratory for immediate physicochemical analysis.

Table 1. Agro-industrial liquid wastes collected.

Raw Material	Collected Waste	Identification Code
Cashew fruit	Cashew apple juice	JPC
Cassava tuber	Water from fermented cassava tuber paste	EFM
Mango	ripe mango pulp	CM

summarizes the sampled effluents.

2.2. Physicochemical Characterization of Agro-Industrial Effluents

Physicochemical parameters, including pH, organic matter (OM), total carbon (Ct), total nitrogen (Nt), total protein, total sugar, and mineral composition, were analyzed in triplicate using standard procedures established by the Laboratory of Industrial Processes, Environmental Synthesis and New Energies (LAPISEN) at the Institut National Polytechnique Félix Houphouët-Boigny (INP-HB), Côte d’Ivoire.

Analyses were conducted using validated reference methods [24,25,26,27,28,29,30,31]. Mineral elements (Na, K, Ca, Mg, Fe, Cu, Mn, Zn, and P) were quantified via atomic absorption spectrophotometry (AAS) as described by Sanchis and Ruiu [25,26]. Results are presented as mean ± standard deviation and expressed on a dry matter basis. A summary of analytical methods is presented in

Table 2. Analytical methods used for physicochemical parameters.

Parameter	Method	Reference
Relative humidity	Gravimetric method	[32]
pH	Electrometric method	[33]
Total carbon (Ct)	Walkley–Black method	[34]
Total nitrogen (Nt)	Colorimetric method	[34]
Total proteins	Biuret colorimetric method	[35]
Organic matter (OM)	AOAC((Association of Official Analytical Collaboration) method	[36]
Dry matter (DM)	—	—
Volatile solids (VSs)	—	—
Ash	—	—
Fiber	Organic matter characterization method	[37]
Density	Measured by weighing in a graduated cylinder	[38]
Minerals	Atomic absorption spectrometry (AAS)	[37]

.

2.3. Preparation of Fermentation Substrates

Each effluent was first filtered through a 5 mm filter paper to remove coarse particulates. Subsequently, 0.5 L of filtrate was diluted with 0.5 L of sterile distilled water, and the mixture was stirred at 500 rpm for 2 min using a magnetic stirrer. The mixed solutions were sterilized in 1 L borosilicate flasks by autoclaving at 121 °C for 30 min and cooled to room temperature before inoculation.

2.4. Bacterial Strain and Inoculum Preparation

The bacterial strain B. thuringiensis var. kurstaki HD-1 (ATCC 33679, HD-1-S-1971 [39]) was obtained from the Canadian Forest Service Laboratory (Sainte-Foy, QC, Canada) through the Bioconversion Laboratory for Wastewater and Sludge Valorization at the Institut National de la Recherche Scientifique—Centre Eau Terre Environnement (INRS-ETE), Université du Québec.

Inoculum preparation followed the method of Vu et al. [40] and Yapo et al. [41]. All culture media (JPC, EFM, CM, and the reference Tryptone Soy Broth—TSB) were adjusted to pH 7.0 using 1 N HCl or 1 N NaOH. TSB medium was autoclaved at 121 °C for 20 min, while effluent-based substrates were autoclaved for 30 min.

A single colony from a pure culture of B. thuringiensis var. kurstaki HD-1 was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of TSB and incubated at 30 °C with shaking at 300 rpm for 8–12 h. Two milliliters (2% v/v) of this pre-culture were subsequently transferred into 500 mL flasks containing 100 mL of each sterilized effluent substrate and incubated under identical conditions for 8–12 h to produce the inoculum used for fermentation.

2.5. Submerged Fermentation Process

Fermentation was conducted in a 3 L bioreactor equipped with a programmable logic controller (PLC) system to regulate temperature, pH, aeration, agitation, and dissolved oxygen (DO). One liter of each sterilized medium, adjusted to pH 7.0, was introduced into the reactor and supplemented with 2% (v/v) sterile olive oil as an antifoaming agent. The medium was then inoculated with 2% (v/v) of the prepared Btk HD-1 pre-culture under aseptic conditions [42].

Fermentation parameters were set at temperature = 30 °C, aeration rate = 0.2–0.4 L air min⁻¹, and agitation speed = 200–250 rpm to maintain DO above 30%. The pH was automatically maintained at 7.0 throughout the fermentation process. Samples were collected at 3 h intervals during the first 24 h, then every 6 h thereafter for up to 48 h. The whole culture was then acidified to pH 5.0 and stored at 4 °C for subsequent analysis [42].

2.6. Macroscopic Observation of Colony Morphology of B. thuringiensis var. kurstaki HD-1 Following the Fermentation Media

To assess the influence of culture media on colony characteristics of Btk HD-1, 100 µL aliquots of whole cultures (TSB, CM, JPC, and EFM) were spread onto trypticase soy agar (TSA) plates (90 mm diameter). For each fermented medium, three replicate plates were prepared and incubated inverted at 30 °C for 16 h. Colony morphology was evaluated using standardized criteria, including colony diameter, color, elevation, margin, and surface texture (as described in Section 3.4). These observations provide insights into how substrate composition affects the macroscopic growth characteristics of Btk HD-1, suggesting underlying physiological adaptations to different nutrient environments.

2.7. Enumeration of Viable Cells and Spores

A total of 36 samples (9 per medium, including the TSB control) were taken at fermentation times of 3 h, 6 h, 9 h, 12 h, 24 h, 30 h, 36 h, 42 h, and 48 h for quantification of viable cells and spores.

The direct plating technique on agar medium was performed by making successive decimal dilutions of the sample to determine the total number of viable bacteria and spores of Btk HD-1 in the supernatant and pellet of the whole culture. For the enumeration of Btk HD-1 colonies, 0.5 mL of the corresponding sample was diluted in sterile glass flasks containing 4.5 mL of pre-sterilized saline water (0.85% NaCl). This suspension was thoroughly mixed using a vortex, and 0.5 mL was transferred to another flask containing the same amount of saline water, i.e., 4.5 mL. The same operation was repeated 12 times to dilute the stock suspension. An aliquot of 0.1 mL from the last 4 tubes was spread on solid TSA (trypticase soy agar) medium in 90 mm diameter Petri dishes. The spreads were performed with a Pasteur pipette in a rake pattern in 4 Petri dishes for the last 4 dilutions. The dishes were incubated upside down in an oven at 30 °C for 20 h. Only results between 30 and 300 colonies were retained to evaluate the total number of viable cells per mL (CFU/mL) [43].

For enumeration of viable spores, the last four dilutions used for total cell counts were subjected to heat shock treatment. Specifically, 1 mL aliquots of each dilution were transferred to sterile glass test tubes (16 × 100 mm) and placed in a precision water bath (Memmert WNB 14, Schwabach, Germany) preheated to and maintained at 65 ± 0.5 °C. Temperature was continuously monitored using a calibrated mercury thermometer (certified accuracy ±0.1 °C) immersed in a control tube containing 1 mL of sterile saline water. Samples were heat-treated for exactly 15 min after temperature equilibration (determined in preliminary tests to be achieved within 45 s), with timing initiated only after all tubes reached 65 °C.

The water bath was calibrated prior to each use against an NIST-traceable reference thermometer. Following heat treatment, the tubes were immediately transferred to an ice-water bath and cooled for 5 min to terminate the heat shock. Serial dilutions were prepared using sterile saline solution (NaCl 0.85%) maintained at 4 °C.

From each heat-treated dilution, 100 µL aliquots were plated in quadruplicate onto TSA plates. Plates were incubated in an inverted position at 30 ± 1 °C for 20 h in a calibrated incubator (Binder BD 53, Tuttlingen, Germany). Colony counts were performed using a digital colony counter (Stuart SC6PL, Stone, UK), and counts between 30 and 300 colonies were retained. The number of colonies counted was multiplied by 2 to return it to 1mL, and the volume of inoculum deposited was multiplied by 10 to return it to 1mL. Finally, spore concentrations were calculated as CFU mL⁻¹ using the following formula [43,44,45]:

CFU mL⁻¹ = number of colonies × (volume of deposited inoculum (mL))⁻¹ × dilution factor

(1)

2.8. Quantification of Bioactive Lipopeptides

To quantify the production of bioactive compounds (iturins and surfactins), 25 mL samples were taken from each culture at 3 h and 48 h of fermentation. Samples were centrifuged, and supernatants were analyzed using a Waters Alliance HPLC system (Waters Corporation, Milford, USA) equipped with a reverse-phase C18 column (5 µm, 250 × 4.6 mm) and UV detection. The mobile phase consisted of a mixture of water, acetonitrile, and trifluoroacetic acid, delivered at 1 mL min⁻¹. Results were expressed in mg L⁻¹.

2.9. Phytopathogenic Agent Used in Bioefficacy Tests

S. rolfsii was used to evaluate the bioefficacy of our whole culture of B. thuringiensis var. kurstaki HD-1 in the laboratory. This phytopathogenic agent was isolated from the main stem of a tomato plant by the National Center for Agronomic Research (CNRA). Pure cultures of S. rolfsii were maintained in the mycotheca at 24 ± 1 °C. This phytopathogenic agent grows rapidly on PDA medium (4 days). Its mycelium is white and thick, lining the surface of the culture medium. The distinctive feature lies in the abundant production of spherical sclerotia. These structures, initially white, progressively turn light brown and then dark brown at maturity. This color change occurs within only 3 days after production. They are distributed on the colony’s periphery or surface. Under the microscope, the hyphae appear hyaline, septate, and highly branched. Unlike certain fungi, S. rolfsii does not produce conidia. Its reproduction relies on sclerotia, true survival organs allowing it to persist in the soil for several cropping seasons (Figure 1).

2.10. Preparation of B. thuringiensis var. kurstaki HD-1 Extracts for Efficacy Testing

At the end of the microscopic characterization (quantity of viable spores) and bioactive compound analysis (surfactins and iturins), the fermented ripe mango pulp (CM) and cassava water (EFM) media were used according to their performance as test controls for the continuation of effectiveness tests in comparison with the fermented conventional medium Tryptone Soy Broth (TSB) used as a control (positive control).

Dilutions of these treatments were prepared in sterile Eppendorf tubes with sterile distilled water, following the recommended doses of reference commercial fungicides, notably Ivory and Callicuivre. The concentrations of the different treatments are registered in

Table 3. Concentrations of the different treatments evaluated.

Treatment Type	ID	Concentrations
Test controls	Btk HD-1/CM	10% (v/v)
		15% (v/v)
		20% (v/v)
	Btk HD-1/EFM	10% (v/v)
		15% (v/v)
		20% (v/v)
Positive control	Btk HD-1/TSB	10% (v/v)
		15% (v/v)
		20% (v/v)

.

The initial solutions of the test treatments were diluted to 80%, 85%, and 90% with sterile distilled water, yielding concentrations of 10, 15, and 20% (v/v).

2.11. Evaluation of the Antifungal Effect of Treatments

Methods of soaking sclerotia in the different treatments and incorporating them into the culture medium, Potatoes Destrose Agar (PDA), were used.

-

Method of incorporating

For each treatment, the doses listed in Table 3 were incorporated into a sterile melted PDA medium to obtain a final volume of 60 mL, which was then divided into four 90 mm Petri dishes (15 mL of PDA medium per dish). After solidification, 5 mm mycelial discs of S. rolfsii, taken from pure cultures aged 7 days on PDA medium, were deposited in the center of the Petri dish.

Each treatment was repeated three times. Two independent series of 36 Petri dishes were conducted, for a total of 72.

-

Soaking method

A total of 10 mL of each treatment, as indicated in Table 3, was used.

Sclerotia collected on pure cultures of S. rolfsii were immersed in these solutions for 24 h, then inoculated on untreated PDA medium. After 24 h, mycelial growth was observed and measured for 7 days.

Each treatment was repeated three times. Two series of 36 Petri dishes were conducted, for a total of 72.

2.12. Evaluation of the Mean Diameter of Mycelial Growth and the Inhibition Rate of S. rolfsii According to the Treatments

Measurements were taken with a graduated ruler every 24 h along two perpendicular axes drawn on the base of each Petri dish, intersecting at the center of the explant (Figure 2) [46]. The average mycelial growth (Dm) was evaluated by the formula below:

$$\mathrm{D}\mathrm{m}\left(\mathrm{c}\mathrm{m}\right)={\displaystyle \frac{\mathrm{L}1+\mathrm{L}2}{2}}$$

(2)

E denotes the explant of known average diameter (X) at time t0; L1 denotes the diameter of explant E according to axis y at time t1, and L2 denotes the diameter of explant E according to axis x at time t1.

The rate of inhibition of mycelial growth (Ic) of S. rolfsii (Ic), depending on the treatment methods (incorporating and soaking) and types of whole culture, was determined by the following formula [47]:

$$\mathrm{I}\mathrm{c}\left(\mathrm{\%}\right)=\left({\displaystyle \frac{\mathrm{D}\mathrm{o}-\mathrm{D}\mathrm{c}}{\mathrm{D}\mathrm{o}}}\right)\times 100$$

(3)

where Do (cm) is the average mycelial growth of S. rolfsii on the PDA culture medium without applied treatment (Control), and Dc (cm) refers to the average mycelial growth of S. rolfsii on the PDA culture medium with application of the treatments.

Also, a comparison of the inhibition rates of fermented alternative media (Btk HD-1/CM and Btk HD-1/EFM) was made with the fermented reference medium (Btk HD-1/TSB) to evaluate the effect of fermentation media on the antifungal properties of Btk HD-1 against S. rolfsii.

2.13. Statistical Analyses

All physicochemical, microbiological, and biochemical data were subjected to one-way analysis of variance (ANOVA) after verifying the homogeneity of variances. When significant differences were detected, Fisher’s LSD test was applied at the 5% significance level. Pearson correlation coefficients were calculated to assess relationships between cell growth and sporulation. Additionally, hierarchical cluster analysis (HCA) was performed to classify agro-industrial effluents based on their physicochemical profiles. The efficacy test data were recorded and processed in Excel, and statistical analyses to evaluate and compare the antifungal effects of our treatments across method, substrate type, and tested doses were conducted using XLSTAT 2014.5.03. All data were tested for normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test) prior to analysis. One-way ANOVA followed by Fisher’s LSD post hoc test (α = 0.05) was used for multiple comparisons. For comparisons involving two factors (e.g., medium type × concentration), two-way ANOVA was performed. Pearson correlation coefficients were calculated to assess relationships between variables. All statistical analyses were performed using XLSTAT 2014.5.03 (Addinsoft, Paris, France) and R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. Physicochemical Characteristics of Agro-Industrial Effluents

The physicochemical profile of the agro-industrial effluents revealed marked variability among the tested substrates, as presented in

Table 4. Physico-chemical characteristics of alternative culture media.

Raw Material	pH	RH (%)	St (%)	MSV (%)	Ct (%)	Nt (%)	C/N	MS (%)	Ash (%)	OM (%)	Fiber (%)	Total Protein (%)
Cashew apple juice (JPC)	4.10 ± 0.05	98.66 ± 0.21	5.60 ± 0.32	76.41 ± 1.24	1.56 ± 0.08	0.28 ± 0.02	5.57 ± 0.41	1.34 ± 0.11	23.59 ± 1.18	2.69 ± 0.14	0.00 ± 0.00	0.78 ± 0.06
Fermented cassava paste water (EFM)	3.80 ± 0.04	90.84 ± 0.35	10.40 ± 0.45	99.86 ± 0.08	2.63 ± 0.11	0.98 ± 0.04	2.68 ± 0.18	9.16 ± 0.23	0.14 ± 0.02	4.53 ± 0.19	0.00 ± 0.00	0.68 ± 0.05
Ripe mango pulp (CM)	4.50 ± 0.06	6.82 ± 0.18	46.00 ± 1.52	79.65 ± 1.37	54.68 ± 1.89	0.70 ± 0.03	78.12 ± 3.45	93.18 ± 1.45	20.35 ± 0.98	94.05 ± 2.11	7.55 ± 0.42	4.74 ± 0.21

pH = hydrogen potential; RH = relative humidity; St = total sugars; MSV = volatile dry matter; Ct = total carbon; Nt = total nitrogen; C/N = carbon/nitrogen ratio; MS = dry matter; Ash = ash content; OM = organic matter.

and

Table 5. Mineral composition of evaluated agro-industrial wastes (mg/kg dry matter).

Raw Material	Na	Mg	P	K	Ca	Mn	Fe	Cu	Zn
Cashew apple juice (JPC)	0.11 ± 0.01	0.00 ± 0.00	0.01 ± 0.002	0.05 ± 0.01	0.20 ± 0.02	0.01 ± 0.001	0.02 ± 0.003	0.02 ± 0.002	0.26 ± 0.03
Cassava paste water (EFM)	0.14 ± 0.02	0.00 ± 0.00	0.01 ± 0.001	0.04 ± 0.01	0.22 ± 0.02	0.00 ± 0.00	0.01 ± 0.001	0.03 ± 0.003	0.17 ± 0.02
Ripe mango pulp (CM)	0.13 ± 0.02	0.00 ± 0.00	0.02 ± 0.003	0.07 ± 0.02	0.19 ± 0.02	0.02 ± 0.002	0.02 ± 0.002	0.01 ± 0.001	0.20 ± 0.02

Na = Sodium; Mg = Magnesium; P = Phosphor; K = Potassium; Ca = Calcium; Mn = Manganese; Fe = Iron; Cu = copper; Zn = Zinc.

. All liquid residues exhibited acidic pH values ranging from 3.80 to 4.50 (Table 4). Among the substrates, ripe mango pulp (CM) had the highest organic matter (OM) content (94.05%) and the highest carbon-to-nitrogen (C/N) ratio (78.12), indicating its richness in carbon-based compounds. In contrast, the fermented cassava paste water (EFM) and cashew apple juice (JPC) displayed lower OM contents and comparable total protein levels across all media.

The mineral composition of the effluents is summarized in Table 5. The concentrations of mineral elements varied from 0.01 to 0.26 mg kg⁻¹ (dry weight basis) depending on the substrate. Zinc (0.17–0.26 mg kg⁻¹), calcium (0.19-0.22 mg kg⁻¹), and sodium (0.11–0.14 mg kg⁻¹) were the most abundant minerals, while phosphorus, iron, copper, and potassium occurred at relatively lower levels (0.01–0.07 mg kg⁻¹). None of the analyzed effluents contained detectable levels of magnesium (0 mg kg⁻¹).

These results indicate that, despite their overall acidity and moderate mineral content, the evaluated agro-industrial residues—particularly mango pulp juice—possess sufficient organic and nutrient potential to support microbial growth and serve as alternative substrates for B. thuringiensis fermentation.

3.2. Hierarchical Cluster Analysis of Agro-Industrial Effluents

Hierarchical cluster analysis (HCA) was performed to assess the degree of similarity among the physicochemical profiles of the evaluated agro-industrial effluents. Effluents grouped within the same cluster exhibited comparable physicochemical characteristics. The analysis distinguished two clearly separated clusters (Figure 3).

Cluster I consisted exclusively of the ripe mango pulp (CM), which was characterized by a high carbon-to-nitrogen (C/N) ratio, elevated levels of total sugars (St), organic matter (OM), and total proteins (Pt), but relatively low concentrations of mineral elements such as manganese (Mn), iron (Fe), copper (Cu), and phosphorus (P).

In contrast, Cluster II included the fermented cassava paste water (EFM) and cashew apple juice (JPC). These substrates were defined by low C/N ratios, reduced contents of total sugars, organic matter, and total proteins, and generally low mineral concentrations.

This classification highlights the distinct nutritional composition of the mango pulp medium compared to other agro-industrial effluents, suggesting its superior potential as a carbon-rich substrate for the growth and metabolite production of B. thuringiensis var. kurstaki HD-1.

3.3. Total Viable Cell and Spore Counts During Fermentation

The dynamics of total viable cells and spores of B. thuringiensis var. kurstaki HD-1 (Btk HD-1) during fermentation are illustrated in Figure 4. Overall, the growth curves displayed similar patterns across all tested media, consisting of four distinct phases (Figure 4a,c,d), except for the ripe mango pulp medium (CM), which exhibited three phases (Figure 4b).

The initial phase (0–9 h) corresponded to a lag period characterized by limited cell production and a slow population increase. During this phase, the highest cell quantity was observed in CM (2.03 × 10¹³ CFU mL⁻¹) and the lowest, 1.32 × 10¹³ CFU mL⁻¹, in JPC and TSB (Figure 4b–d). Following this lag phase, rapid exponential growth occurred up to 30 h of fermentation.

During the exponential phase, total cell concentrations increased significantly in the cashew apple juice medium (JPC), thereby supporting the highest vegetative cell biomass among all tested substrates (from 1.32 × 10¹³ to 8.29 × 10¹³ CFU mL⁻¹; Figure 4d).

Similarly, the spore production graphs (Figure 4a,c,d) followed four distinct phases, with maximum sporulation observed around 30 h, except in the CM medium (Figure 4b), which reached its peak earlier. The early fermentation period (0–9 h in CM; 0–12 h in JPC; and 0–24 h in EFM and TSB) corresponded to slow spore formation, followed by a sharp increase that reached its maximum at 30 h in most media. In the reference TSB, however, the sporulation peak was reached at 24 h. Thereafter, spore counts declined, then stabilized between 36 h and 48 h.

Among all substrates, CM induced the highest spore density (1.46 × 10¹³ CFU mL⁻¹), followed by JPC and EFM, whereas the lowest sporulation rate (3.34 × 10¹² CFU mL⁻¹) was recorded in TSB.

Overall, both CM and JPC media outperformed the conventional TSB medium by supporting higher biomass accumulation and enhanced sporulation in the bioreactor. A significant positive correlation (r = 0.418; p < 0.05) was observed between mean cell counts and spore production, indicating that sporulation was closely associated with bacterial growth during fermentation.

3.4. Influence of Culture Media on the Colony Morphology of B. thuringiensis var. kurstaki HD-1

Colony morphology was assessed using standardized criteria including: (i) colony diameter (measured using ImageJ software v1.53, NIH, USA) from 10 randomly selected colonies per plate; (ii) colony color (referenced against the Munsell color chart); (iii) colony elevation (flat, raised, convex); (iv) colony margin (entire, undulate, filamentous); and (v) surface texture (smooth, rough, wrinkled).

Colonies grown on EFM and TSB exhibited similar characteristics: mean diameter 2.3 ± 0.4 mm, raised elevation, undulate margins, rough surface texture, and yellowish coloration (Munsell 2.5Y 8/10). Water droplets were observed on the reverse side of plates in 85% of EFM and 92% of TSB cultures (Figure 5a,c).

In contrast, colonies on JPC and CM media were significantly larger (mean diameter 4.7 ± 0.6 mm; p < 0.05), with flat elevations, entire margins, a smooth, glossy surface, and bright, whitish coloration (Munsell 5Y 9/1). No condensation droplets were observed on the reverse sides of these plates (Figure 5b,d).

These observations suggest that substrate composition significantly influences the macroscopic growth characteristics of B. thuringiensis var. kurstaki HD-1, likely due to variations in available nutrients and moisture dynamics among the fermentation media.

3.5. Production of Iturin and Surfactin

The concentrations of iturin and surfactin produced by B. thuringiensis var. kurstaki HD-1 (Btk HD-1) varied depending on both the fermentation substrate and duration of fermentation. Generally, the levels of these bioactive lipopeptides increased progressively with increasing fermentation time (Figure 6).

• Limit of quantification (LOQ) for iturin: 5 mg/L; measurement uncertainty: 2%.
• Limit of quantification (LOQ) for surfactin: 15 mg/L; measurement uncertainty: 1.5%.

Among all tested media, the ripe mango pulp (CM) supported the highest synthesis of iturin and surfactin, surpassing that observed in the conventional Tryptone Soy Broth (TSB) medium. In CM, iturin concentrations increased from 37.7 to 102.2 mg L⁻¹, and surfactin concentrations rose from 237.8 to 554.7 mg L⁻¹ between 3 h and 48 h of fermentation (Figure 6a,b). By comparison, in TSB, iturin and surfactin levels increased from 15.9 to 56.6 mg L⁻¹ and from 220.2 to 389.6 mg L⁻¹, respectively, over the same period.

Conversely, the cashew apple juice (JPC) and fermented cassava paste water (EFM) media supported lower production levels of both metabolites. The lowest concentrations were recorded in EFM, ranging from 7.4 to 39.4 mg L⁻¹ for iturins and from 87.7 to 107.6 mg L⁻¹ for surfactins.

Overall, surfactin production consistently exceeded iturin production across all media. The surfactin yield was approximately twofold higher than iturin in JPC and EFM media, and up to fivefold higher in CM medium. These results highlight the strong influence of carbon- and nitrogen-rich substrates, particularly mango pulp juice, on lipopeptide biosynthesis by B. thuringiensis during submerged fermentation.

3.6. Antifungal Activity of B. thuringiensis var. kurstaki HD-1 Against S. rolfsii

The antifungal activities of our fermented ripe mango pulp and fermented cassava water, based on B. thuringiensis var. kurstaki HD-1, were evaluated and compared with that obtained from the conventional culture medium TSB using two methods: (i) incorporation of treatments into PDA medium (assessing direct growth inhibition), and (ii) soaking of sclerotia in treatments prior to plating on untreated PDA (assessing preventive activity). Figure 7 reveals the absence of a significant difference between the fermented ripe mango pulp (CMBt), fermented cassava water (EFMBt), and the fermented conventional tryptone soy broth medium (TSBBt), suggesting that B. thuringiensis is capable of producing antifungal metabolites effective against S. rolfsii regardless of the nature of the fermentation substrate. The comparable effectiveness of B. thuringiensis var. kurstaki HD-1 obtained by fermentation in our alternative substrates reinforces the interest in their use in low-cost, environmentally friendly biocontrol strategies. The exploitation of agro-food residues such as fermented cassava water or ripe mango pulp is part of a circular economy and waste valorization approach, particularly suited to the agricultural contexts of tropical countries like Côte d’Ivoire. Also, no significant dose–response relationship was observed between the concentrations of 10%, 15%, and 20% of the whole culture tested. This result could be explained by the achievement, from the lowest tested concentration, of an inhibitory threshold sufficient to exert a maximal antifungal effect on S. rolfsii. Similar observations have been reported for metabolites of Bacillus spp. whose antifungal activity relies on rapid membrane mechanisms, limiting the appearance of a response gradient at higher doses.

4. Discussion

The physicochemical analyses revealed that all tested culture media exhibited acidic pH values but differed markedly in their organic matter, protein, total sugar, and carbon-to-nitrogen (C/N) ratios. In contrast, their mineral composition profiles were relatively similar. This variability in nutrient composition, combined with fermentation parameters and conditions, significantly influenced cell growth, sporulation, and the biosynthesis of bioactive lipopeptides such as iturins and surfactins. Previous studies have demonstrated that minerals, including zinc, iron, manganese, magnesium, and calcium, as well as carbon and nitrogen sources, play crucial roles in the growth, sporulation, and metabolite synthesis of B. thuringiensis [48,49].

Starch-processing wastewater from agro-industrial facilities is currently considered one of the most promising low-cost substrates for Bt production through bioconversion. Several investigations have successfully demonstrated its suitability for large-scale fermentation of B. thuringiensis [15,50]. In the present study, the nutrient potential of the evaluated agro-industrial effluents—fermented cassava paste water (EFM), cashew apple juice (JPC), and ripe mango pulp (CM)—was found to be equally favorable for the cultivation and bioactive metabolite production of B. thuringiensis var. kurstaki HD-1.

Among these, the mango pulp medium (CM) distinguished itself by its high levels of organic matter and total sugars, reflected in its elevated C/N ratio. The richness of mango residues in carbohydrates has also been documented by Awodi et al. [51]. This composition likely contributed to CM’s superior performance in supporting both vegetative growth and sporulation, as well as in promoting the synthesis of bioactive molecules. Similar findings were reported by Içgen et al. [52], Liu et al. [53], and Zouari and Jaoua [54], who emphasized that spore and crystal formation in B. thuringiensis is tightly regulated by the carbon-to-nitrogen balance in the culture medium. Likewise, Nickerson and Bulla [55] demonstrated that the absence of carbon sources inhibits sporulation, while Yang and Wang [56] showed that the depletion of both organic and inorganic nitrogen sources triggers the synthesis of toxin proteins and initiates sporulation.

The morphological and microbiological diversity observed across the tested media supports previous observations by Farrera et al. [57], who reported that changes in carbon sources can alter B. thuringiensis growth patterns and crystal morphology. The pronounced differences among media highlight, on one hand, the importance of balanced carbon and nitrogen availability for sustaining exponential growth and, on the other hand, their influence on the induction of regulatory pathways controlling sporulation and δ-endotoxin (Cry protein) synthesis [58].

In this study, exponential growth peaked before 24 h of fermentation, followed by a stationary phase between 24 and 30 h and a post-sporulation lysis phase between 36 and 48 h, during which spores and crystal proteins were released [59]. The moderate positive correlation (r = 0.418; p < 0.05) observed between mean cell counts and spore production suggests that, while biomass accumulation partly determines sporulation potential, the transition to sporulation is also governed by specific regulatory mechanisms independent of cell density. This observation aligns with established models for Bacillus species, in which sporulation is controlled by a cascade of regulatory events triggered by environmental cues and quorum-sensing signals.

Therefore, optimizing culture conditions to maximize sporulation and bioactive metabolite production must consider not only bacterial growth parameters but also environmental and physiological factors that specifically regulate the sporogenic process.

The results of efficacy tests confirm the antifungal activity of B. thuringiensis against S. rolfsii, a telluric phytopathogenic fungus responsible for significant economic losses on many food and market gardening crops [60]. Although B. thuringiensis is historically recognized for its insecticidal properties associated with Cry proteins, several studies have demonstrated its ability to inhibit phytopathogenic fungi by producing bioactive secondary metabolites [61,62].

The significant and stable inhibition of mycelial growth observed over the 4 days of incubation suggests a direct interaction between the compounds produced by B. thuringiensis and the cellular structures of S. rolfsii, as reported for other Bacillus species [63]. The observed antifungal activity could be attributed to the production of cyclic lipopeptides such as iturins, fengycins, and surfactins, which are widely reported in B. thuringiensis, and related species such as B. subtilis and B. amyloliquefaciens [62,63].

These compounds are known to interact with sterol-rich fungal cell membranes, causing membrane permeabilization, leakage of intracellular constituents, and inhibition of mycelial elongation [64].

The absence of a significant difference between the fermented ripe mango pulps, cassava water, and tryptone soy broth of B. thuringiensis indicates that the nature of the fermentation substrate did not influence the antifungal efficacy of B. thuringiensis against S. rolfsii. This result suggests a strong metabolic capacity in the strain used, enabling it to synthesize active antifungal metabolites in both conventional and alternative media. This metabolic capacity has already been described in several species of the genus Bacillus, capable of synthesizing antifungal lipopeptides in both synthetic and complex media, as well as in media derived from agri-food residues. The observed antifungal activity could be attributed to the production of cyclic lipopeptides, such as iturins, fengycins, and surfactins, which are widely reported in B. thuringiensis. These compounds are known to disrupt the integrity of the fungal membrane, inducing cell constituent leakage and inhibition of mycelial growth.

Previous studies have shown that Bacillus spp. can produce antifungal lipopeptides in a wide range of substrates, including complex media and agri-food residues, without major modification of their biological activity [64,65]. Thus, alternative media based on fermented cassava water or ripe mango appear to provide sufficient carbon and nitrogen sources to support the biosynthesis of these bioactive compounds.

No significant dose–response relationship was observed between the concentrations of 10%, 15%, and 20% of the whole culture incorporated into PDA medium. This result could be explained by rapid attainment of the minimum inhibitory concentration (MIC) at the lowest concentration tested, resulting in a maximal antifungal effect that is not proportional to increasing dose.

Similar observations have been reported for lipopeptides produced by Bacillus spp., whose antifungal activity relies on membrane perturbation mechanisms leading to rapid inhibition of fungal growth [66,67]. In this context, increasing the concentration beyond the effective threshold does not necessarily lead to additional measurable inhibition, particularly on a rich solid medium such as PDA.

5. Conclusions

This study demonstrated the potential of agro-industrial effluents as alternative nutrient media capable of supporting the growth, sporulation, and production of bioactive compounds by B. thuringiensis var. kurstaki HD-1. The nutritional profiling of the tested substrates revealed two distinct classes based on their mineral composition and physicochemical properties.

Enumeration of viable cells and spores, together with the quantification of secondary metabolites, confirmed that the alternative media—ripe mango pulp (CM), cashew apple juice (JPC), and fermented cassava paste water (EFM)—were competitive with the conventional Tryptone Soy Broth (TSB) medium. Among these, CM exhibited the highest sporulation rates and the greatest biosynthesis of bioactive metabolites, particularly iturins and surfactins. These lipopeptides confer antifungal and antibacterial properties to B. thuringiensis, making it a promising biological control agent for tomato pathogens and pests.

The valorization of such agro-industrial residues not only supports sustainable waste management but also offers a cost-effective strategy for eco-friendly biopesticide production within a circular bioeconomy framework.

The comparable effectiveness of B. thuringiensis var. kurstaki HD-1 products fermented in alternative substrates and in classical laboratory media highlights their strong potential for the development of economically accessible biocontrol solutions. The use of agro-food residues, such as fermented cassava water and ripe mango pulp, is part of a circular economy approach to waste recovery, in line with the principles of agroecology.

These results are particularly relevant for tropical agricultural systems, where access to synthetic inputs is limited and soil-borne diseases such as those caused by S. rolfsii represent a major constraint on productivity.