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Biopesticide production using Bacillus thuringiensis kurstaki by valorization of starch industry wastewater and effluent from aerobic, anaerobic digestion



Unconventional alternatives such as aerobic and anaerobic effluent from starch industry contain essential nutrients for Btk active ingredient synthesis. Effluent from starch industry is rich in carbon and nitrogen and can replace expensive feedstock used during the fermentation process.


The main objective of this study was to achieve a biopesticide formulation from starch industry wastewater (SIW) with high entomotoxicity (UI/ml) of larvae comparable to Foray 76B, which is a commercial biopesticide.


Bacillus thuringiensis var kurstaki HD1 (Btk) strain was cultivated and sub-cultured to aerobic, anaerobic digested effluent and SIW. Pre-treatment was carried on these different substrates to enhance the residual carbon required for Btk growth and delta endotoxin synthesis. After 48 hours of fermentation, cells count and delta-endotoxin were determined. A biopesticide formulation containing fermented broth and adjuvants was fed to larvae to determine larvae mortality.


Btk cell growth and sporulation profile in SIW media displayed a high total cell count and viable spores compared to btk growth in anaerobic or aerobic media after 48h fermentation. The maximum endotoxin concentration in the SIW medium was 435μg/mL, whereas, in anaerobic and aerobic effluent, the maximum concentrations were at 161 μg/mL and 136 μg/mL, respectively. When acidic treatment was performed at pH 2 for these substrates, entomotoxicity obtained from aerobic and anaerobic biopesticide formulations displayed significantly higher entomoxicity than the untreated ones. The entomotoxicity of SIW treated at pH 2 was equivalent to the standard Foray 76B which is 20,000 IU/μL.


Anaerobic and aerobic effluent did not contain enough total organic carbon to augment Btk growth and entomotoxicity. Substrates pre-treated at pH 2 provided significant organic matter for Btk growth and resulted in larval mortality equivalent to the com ercial biopesticide Foray 76B.


A significant demographic growth rates has led to an unselective use of chemical pesticides to protect crops for high productivity. The drawbacks of applying chemical pesticides are pollution of groundwater, cultures, and environmental pollution (Marrone, 1999). In spite of these disadvantages, the use of chemical pesticides is still in use. The cost and convenience of using them are plausible explanations. Alternatives exist, such as biological pesticides based on Bacillus thuringiensis (Bt) microorganisms, whose endotoxin protein crystals are lethal to many lepidopteran, coleopteran and dipteran pests. Bt-based biopesticides account for 97% of the world market for biopesticides [1, 2]. Bacillus thuringiensis is a gram-positive, aerobic bacterium. This strain is capable of sporulating and concomitant parasporal crystal synthesis during their growth. The insecticidal activity of Btk arises from the presence of viable spores and endotoxins (Cry IAb). The insecticidal activities of Btk endotoxin against lepidopteran and diptera (flies and mosquitoes and beetles) species were reported [3]. Bacillus thuringiensis produces protein crystals consisting of endotoxin delta, which bind to receptors on larval epithelial cells [4]. This destabilizes the epithelial cells by creating transmembrane pores in the cell membrane. This affects the integrity of the epithelial cells, resulting in paralysis of the larval digestive system and cessation of feeding. At the same time, viable spores produced during the stationary phase of bacterial growth will germinate in the midgut of the budworm and produce more Bt cells and protein crystals. The proliferation of the bacteria will cause larvae to become septic and die [5]. A biological pesticide is only effective, as long as it has a significant potential impact on the target pest, good performance under varying field conditions, cost effectiveness, and satisfactory end-user return Bt is easily cultivated on a synthetic medium [6]. The inherent cost of commercial medium for fermentation hinders the competitiveness of biopesticides compared to chemical pesticides and is also an obstacle to their commercialization. Thus, the food waste industry stream or wastewater treatment plants are alternatives to synthetic products [7]. Sewage sludge has been successfully used as a feedstock for the production of Bt biopesticides with lower process costs [8, 9]. In the INRS research group, a Bt biopesticide was produced using wastewater from the starch industry (SIW), as it is rich in carbon and nitrogen and can replace expensive feedstock used during the fermentation process [5, 10].

Unconventional alternatives contain essential nutrients for Btk active ingredient synthesis. In [9], it was showed that the use of residues from starch industries provides a higher entomotoxicity than conventional media. Agro-processing residues contain a high concentration of chemical oxygen demand (COD) in the form of carbon and nitrogen sources (starch, gluten, protein, fiber and other minerals) [11]. It represents a severe problem of treatment and final disposal in the environment. Thus, the general practice provides either aerobic [12, 13] or anaerobic treatment [11, 13] of these residues. In the process of treatment of these types of residues, effluent rich in organic matter is generated. These could contain the needed nutrients for Btk growth. This study results demonstrated a potential new substrate derived from the SIW treatment plant digester for biopesticide production. Moreover, the physical parameters relating to the viscosity, zeta potential and particle size of these substrates were analyzed before each fermentation.

Materials and methods

Bacterial microorganism and cultural medium

Bacillus thuringiensis var kurstaki HD1 (Btk) strain was obtained from the Canadian Forest Service laboratory, Ste-Foy, Québec. The Btk strain was cultivated and sub-cultured on Tryptic soy agar (TSA) at 30 °C for 12 h. The grown pure bacterial cultural was stored at 4 °C for inoculum preparation [4, 15].

Growth medium


The culture medium (SIW—starch industry wastewater) for Btk fermentation was obtained from ADM (155 Avenue d'Iberia, Candiac, QC J5R 3H1), a cornstarch processing industry. The composition of this medium, analyzed by ICP-AES axial Vista, is presented in Table 1. Table 2 shows the concentration of total organic carbon (Shimadzu VCPH, NPOC Curve 0—5 mg/L), total organic nitrogen (Shimadzu VCPH, TN Curve 0–5 mg/L) and ammonia nitrogen (Lachat Method) present in this medium. The SIW is stored in the cold room at 4 °C until it is used. Its shelf life is less than two months [4].

Table 1 SIW characteristics—Suggested wavelength and estimated detection limit for minerals selected by ICP-AES axial Vista
Table 2 Physical and chemical characteristics of substrates

Anaerobic (AnaeroD-SIW) and aerobic (AeroD-SIW) effluent

Starch industry wastewater is rich in COD and BOD (above 300 mg/L). The regulations require the treatment of these residual materials before they are discharged into municipal treatment plant pipes. Thus, starch wastewater undergoes first anaerobic digestion (to generate methane) followed by aerobic digestion. Both treatments generate effluent rich in organic matter (significant organic nitrogen source) named secondary anaerobic effluent (AnaeroD-SIW) and secondary aerobic effluent (AeroD-SIW). The characteristics of these substrates are reported in Table 2 [4, 9, 16].


A single colony picked from nutrient agar petri dish containing colonies of Btk was seeded in 50 mL Erlenmeyer flasks containing 1.2 mL of Tryptic soya medium as a Pre-culture 1(Pc1). Pc1 was incubated at 30 °C, pH 7.0 and 200 rpm for 9 h. This seed culture was transferred to 500 mL flasks containing 60 mL (Pc2) of SIW, AeroD-SIW, AnaeroD-SIW. Pc2 was transferred into 5 L bioreactor with 3 L working. To reduce the latence phase at the beginning of fermentation, Pc2 and production medium contain same medium [4].


Fermentation was carried out for 48 h. Every 6 h, samples were taken for total cells and spores count [4]. To maintain a concentration of DO > 30%, the airflow and agitation rate were maintained between 2 and 2.5 L of air per minute and 300–350 rpm, respectively. The temperature was maintained at 30 °C by thermostat supplying cooling water during fermentation. Fermentation media pH was maintained at pH 7 using a computer-controlled operating system. Samples were collected at regular intervals (every 6 h) to determine the total cells and spores count, and delta-endotoxins in fermented broth. At the end of fermentation, the fermented broth's pH was adjusted to 4.5 to eliminate the solubilization of proteins by the proteases active at alkaline pH and to arrest the growth of contaminants [4, 8, 17].

SIW, AeroD-SIW and AnaeroD-SIW pre-treatment at different pH

SIW, AeroD-SIW and AnaerdoD-SIW as substrates are complex media consisting of complex biodegradable materials [4, 17]. These biodegradable materials often require treatment with basic pH (NaOH) or acidic pH (sulphuric acid) to provide high availability of the complex carbon form to microbes [5, 9]. Thus, each media (50 mL SIW or AeroD-SIW or AnaeroD-SIW) was adjusted to different pH, 2 (sulfuric acid); 7(NaOH); 12 (NaOH) and then sterilized at 121 °C, 15 psi pressure for 15 min in an autoclave. The suspended solids concentration obtained by batch centrifugation showed that the treatment at pH 2 resulted in significant solubilization of all substrates (Table 2). The pre-treated samples (pH 2 and 7) were analyzed by LC–MS-MS Thermo TSQ Quantum (Table 3).

Table 3 Carbohydrate determination by LC–MS–MS after substrate pre-treatment at different pH values

Fermentation using the substrates treated at pH 2 was carried out in 5 L bioreactor under the same operating conditions as those specified above. Analysis of Btk total cell counts, viable spores count and endotoxins was performed, and comparison was made with the results obtained by growing Btk without pre-treatment of the substrate [4, 9].

SIW media fortified with AeroD-SIW, AnaeroD-SIW and SIW medium at different concentrations

Substrates from agro-industry residues or wastewater treatment plants were characterized for the presence of nutrients in the form of biosolids or suspended solids [8]. These wastes contain suspended solids, dissolved and particulate (organic carbon, nitrogen, and ammonia). Hence, the AeroD-SIW and AnaeroD-SIW and SIW were concentrated by centrifugation to achieve 30 g/L of SS in effluent. SIW medium represents the most nutrient-rich substrate (Table 2) [4, 9].

SIW medium was fortified by concentrated solids obtained by centrifugation of AeroD-SIW, AnaeroD-SIW and SIW substrate. The centrifuged solids were added to SIW and pre-treated at pH 2 so that the final concentration of SS in each case was 30 g/L. The operating conditions for fermentation were identical to those detailed in the fermentation section. Btk total cells count, viable spores count, and endotoxin concentrations were measured on fermentation samples collected at 3-h intervals during the first 12 h of fermentation and at 6-h intervals up to 48-h fermentation.

Total cells and viable spore count

Total cells and spore counts were determined by counting bacterial colony forming units (CFU) on TSA (Tryptic Soy Agar) plates. All the CFU counts were obtained from the average value of experiments conducted in triplicates. For total spore count, serially diluted samples were placed in a water bath (Buchler Instruments) at 80 °C for 10 min and immediately immersed in ice for 10 min. Then, 0.1 mL of diluted sample was plated on TSA medium and incubated at 30 °C for 24 h (Imperial II Incubator oven) [4, 8]. The presented values are the mean of three separate experiments ± standard deviation (SD).

Endotoxin estimation

Btk produces cry toxins encoded by different cry genes found on Btk plasmids. Those cry toxins consist of parasporal inclusions, which contain crystal proteins or endotoxins. They have a large spectrum of action against larvae of Lepidoptera, Diptera, and Coleoptera [14, 18]. Endotoxin concentration was determined based on cry proteins' solubilization under alkaline conditions [6, 19]. One mL of each sample was centrifuged at 10,000 g for 10 min at 4 °C. The pellet containing the mixture of spores, crystal proteins, and cell debris was used to estimate the soluble insecticidal proteins (delta-endotoxin) in an alkaline medium. The pellets were washed thrice with 1 mL of 0.14 M NaCl and 0.01% Triton X-100 solutions. This washing step aids in removing soluble proteins and proteases, which might stick to the centrifuged pellets and affect the integrity of the crystal proteins [5]. The pellets of crystal proteins were dissolved in 0.05 N NaOH (pH 12.5) for 3 h at 30 °C. After crystal solubilization, the suspension was centrifuged at 10,000 g for 10 min at 4 °C, and the pellet containing the spores and cell debris was discarded. The supernatant containing the insecticidal crystal proteins (soluble) was used to determine the concentration of delta-endotoxin by the Bradford method using bovine serum albumin (BSA) as a protein standard (MM Bradford 1976). The presented values are the mean of three separate experiments ± Standard Deviation (SD).

Zeta potential

Zeta potential (ζ) of each sample was analyzed using Zetaphoremeter IV, Zetacompact Z8000 (CAD instrumentation France) [10]. The zeta potential values were obtained from the average of 3 measurements. The average values are presented with its half-width confidence interval at a 95% confidence level.


An optimal viscosity will provide a uniform distribution of active ingredients on the tree leaves and promote efficient applications [4, 10]. Viscosity was measured using the Brookefield DVII pro, a viscometer with Rheocalc 32, SC-34 pin software (small sample adapter) [4]. The presented values are the mean of three separate experiments ± Standard Deviation (SD).

Particle size

Particle size analysis was carried out using a Horiba "Laser scattering particle size analyzer (LA-950)". The stirrer and recirculation pump speed were kept at 250 and 500 rpm to minimize flocculent particles' breakage. For analysis, each sample was analyzed three times to confirm the validity and reproducibility of the results [4]. This method is based on the principles of Fraunhofer diffraction and Mie scattering [10]. Results obtained are described below:

D50 indicates that 50% of the particle size fall between the measured value.

D90 indicates that 90% of particles are smaller than the obtained value.

D10 indicates that 10% of the particles have a smaller size than the obtained value.

Entomotoxicity bioassay

Entomotoxicity (Tx value) of Btk was estimated by bioassay against third larvae of spruce budworn (Choristoneura fumiferana) following the diet incorporation method of Dulmage and Beegle [20]. The standard commercial preparation of entomotoxicity was used as a reference standard to analyze the TX value. The detailed procedure of bioassay technique was presented in [15]. The Tx value was evaluated by comparing the mortality percentages with dilutions of the samples (suspended pellet as well as whole fermented broth) and these values were against the standard. The presented values represent the mean of three determination of three independent experiments ± SD.

$$\frac{{Entomotoxicity \left( {IU/ \mu L} \right) = \left( {\frac{{\% ( mortality\,\, sample \,\,10^{ - 3} }}{{\% (mortality\,\, standard \,\,10^{ - 3} }}} \right) + \left( {\frac{{\% (mortality\,\, sample \,\,10^{ - 4} }}{{\% (mortality\,\, standard \,\,10^{ - 4} }}} \right) + \left( {\frac{{\% (mortality\,\, sample \,\,10^{ - 5} }}{{\% (mortality \,\,standard \,\,10^{ - 5} }}} \right)}}{3}\times19950.$$

Fortification impact on particle size and fermented broth viscosity

The physical characteristics (particle size, viscosity) of the waste media (Digested effluent, starch wastewater) can have a significant impact on fermentation (oxygen transfer) and formulation (droplet size, viscosity, zeta potential) [4]. The increased suspended solids concentration and extracellular polymeric substances (EPS) present in effluent can affect particle biosorption, flocculation and floc structure [21]. High EPS concentration in sludge (aggregation between bacteria and EPS) can lead to high viscosity affecting fermentation and biopesticide formulation [8].

Therefore, the analysis of particle size, viscosity (Brookefield DVII pro, Rheocalc 32, SC-34) and zeta potential (Zetaphoremeter IV, Zetacompact Z8000) was performed on samples of different substrates (Table 4).

Table 4 Physical parameters of the different substrates studied before fermentation


The fermented broth was centrifuged using a batch centrifuge (Sorval RC5c plus superspeed), and the concentrated slurry was recovered. To obtain a low viscosity of the concentrated product and facilitate easy field application, fermented broth (after 48-h fermentation) and chemical additives were added to the centrifuged slurry [8]. The ingredient additives were prepared separately (Table 5). They were used as follows; carboxymethylcellulose (emulsifier, stabilizer), Xanthan gum (provides better adhesion to the foliage), molasses (adhesion, UV protectant, and phagostimulant) and potassium silicate (combined with sodium acetate buffer) [17]. The fermented broth's homogenized ingredients were mixed with antimicrobial compounds like propionic acid, ascorbic acid, and acetic acid [9]. The mixtures of additives, fermented broth and the centrifuged slurry were homogenized in a large vessel equipped with a stirrer (200 rpm) (Caframo stirrer type Rzr50) [4]. The pH of the formulated product was 5. Samples were collected in sterilized bottles for entomotoxicity analysis. The final product was stored under sterile conditions.

Table 5 Formulation composition for 100L volume

Results and discussion

Btk total cell growth, viable spores and endotoxin synthesis in AeroD-SIW, AnaeroD-SIW and SIW media

Btk cell growth and sporulation profile in SIW media displayed a high total cell count of 2.90 × 108 CFU/mL and viable spores of 2.4 × 108 CFU/mL compared to Btk growth in AnaeroD-SIW or AeroD-SIW after 48 h fermentation (Fig. 1a, b). Figure 1c shows the Btk endotoxin synthesis profile in anaerobic, aerobic and SIW media during 48 h of fermentation. The maximum endotoxin concentration in the SIW medium was 435 µg/mL, whereas, in anaerobic and aerobic effluent, the maximum concentrations were at 161 µg/mL 136 µg/mL, respectively (Fig. 1c). Btk cell growth, sporulation and endotoxin synthesis are mechanisms that require energy in the form of ATP from carbon metabolism through the Krebs cycle and oxidative phosphorylation [22]. Table 2 indicates that total organic carbon (TOC) is significantly lower in anaerobic and aerobic effluent than in SIW. This is because, during anaerobic and aerobic digestion of starch wastewater, microorganisms utilize the substrate (carbon source) present in the medium. Under anaerobic digestion, there is a lower degree of digestion (more carbon present in the effluent) due to the presence of a large proportion of inactive microorganisms (aerophilic microorganisms). The methanogens and sulfate-reducing microorganisms remain active under anaerobic conditions.

Fig. 1

Btk profile employing AeroD-SIW, AnaeroD-SIW and SIW: a total cell count b viable spores count c endotoxin synthesis

Thus, biomass production during aerobic and anaerobic digestion leads to an enrichment of the effluent in nitrogen sources and carbon depletion. Carbon is lost as CO2 or fixed into methane (biogas generated during anaerobic digestion).

Further, reducing sugars analysis by LC–MS–MS at pH 7 shows that the carbohydrates needed for the synthesis of Btk active ingredients were present at a low concentration in various substrates studied (Table 3). Thus, after treatment at pH 2, the glucose concentration is improved a little in SIW substrates, aerobic and anaerobic effluent. The concentration of galactose and fructose available for Btk has improved in AeroD-SIW and SIW substrate. The xylose concentration, a 5-carbon sugar not assimilable by Btk, was improved in the three substrates studied. Though a little, these results may directly impact Btk cell growth, sporulation and endotoxin synthesis in anaerobic, aerobic and SIW media. Btk total cell count and viable spores count were 5.5 × 108 CFU/mL, 4.9 × 108 CFU/mL, respectively, in SIW; 3.5 × 108 CFU/mL, 2.5 × 108 CFU/mL in anaerobic effluent and 1.5 × 108 CFU/mL, 1 × 108 CFU/mL in aerobic effluent after 48 h (Fig. 2a, b). These substrates' pre-treatment was done by pH 2 adjustment and sterilization. Figure 2c shows Btk endotoxin synthesis profile in media pre-treated at pH 2 (i.e., adjusted to pH 2) and then sterilized. Endotoxin concentration was 550 µg/mL in pre-treated SIW medium, 350 µg/mL in pre-treated AneroD-SIW and 200 µg/mL in pre-treated AeroD-SIW. These results confirmed the significant role of assimilable carbon in Btk cell growth, sporulation and endotoxin synthesis. The yield of biomass in function of SIW is 39%; while with Tryptic soybean medium, it is 50%. Indeed, the metabolism of sludge requires a higher energy expense in the production of enzyme to degrade the complex materials to produce biomass.

Fig. 2

Btk profile from AeroD-SIW, AnaeroD-SIW and SIW pre-treated at pH 2: a total cells count b viable spores count c endotoxin synthesis

SIW Fortification by AnaeroD-SIW, AeroD-SIW and SIW

To study the contribution of anaerobic and aerobic effluent rich in ammonical nitrogen and organic nitrogen towards Btk growth, sporulation and endotoxin production, anaerobic effluent, aerobic effluent and SIW were separately centrifuged. The centrifuged solids were supplemented to fresh SIW substrate so that their solids concentration in SIW was 30 g/L and after that subjected to Btk fermentation. The SIW, AeroD-SIW and AnaeroD-SIW solids present in the effluent are homogeneous and of very small particle size (Table 4). When autoclaved at 121 °C for 15 min, a thermal digestion of solids particles going from starch to simple sugar occurs which are easily accessible to bacteria (Table 3). While other solids containing starch and other type of carbon more refractory to degradation require amylase production by Btk for carbon assimilation [8, 16].

The profiles of Btk cell count and spore count obtained in these 3 types of medium are presented in Fig. 3a, 3b. At 48 h, total cells count and viable spores count were 5.6 × 108 CFU/mL, 5 × 108 CFU/mL in SIW fortified with 30 g/L SIW; 5.6 × 108 CFU/mL, 4.5 × 108 CFU/mL in SIW fortified with 30 g/L anaerobic effluent; 4.64 × 108 CFU/mL, 3.5 × 108 CFU/mL in SIW fortified with 30 g/L aerobic effluent. At 48 h, endotoxin concentration was 680 µg/mL, 540 µg/mL, 450 µg/mL, respectively, in SIW fortified with 30 g/L SIW, SIW fortified with 30 g/L anaerobic effluent and SIW fortified with 30 g/L aerobic effluent (Fig. 3c).

Fig. 3

Btk profile using pre-treated SIW at pH 2 and fortified with 30 g/L of AeroD-SIW, AnaeroD-SIW and SIW: a total cells b viable spores c endotoxin synthesis

When compared to 15 g/L fortified SIW medium, these results showed an improvement in cell count, spore count, and endotoxin synthesis in SIW fortified with solids of SIW or solids of aerobic or anaerobic effluent. However, fortifying SIW with 30 g/L solids of SIW, there was no improvement in Btk cell count or spore count (Fig. 3a, 3b), but improved endotoxin synthesis (Fig. 3c) fortified SIW with 15 g/L solids of SIW. In fact, during SIW solids fortification, the supernatant of SIW (not used in fortification) contains a significant fraction of total organic carbon in dissolved form [5]. The fraction of particulate organic carbon, collected in the pellet and used to fortify the medium, does not contain enough nutrients (carbon) to support Btk cell growth, sporulation and endotoxin synthesis. In [23], it was stated that to achieve high cell growth and sporulation, simple sugars (glucose) and proteins (organic forms) are necessary from the beginning and during Btk fermentation. Also, early sporulation, cell growth suppression due to the inadequate nature of nitrogen sources would have resulted in the availability of substrates for endotoxin synthesis. In [24], it was mentioned that the high availability of ATP during Btk sporulation positively affected the production of crystals protein. However, the rheology (non-Newtonian behavior) of effluent [8], the viscosity, the presence of EPS [25], the presence of the carbon fraction in particulate form provide adequate effluent nutrients in the pellet after centrifugation for endotoxin synthesis [15].

Particle size, viscosity and zeta potential in different substrates

Table 4 presents the values of viscosity, particle size and zeta potential of the different substrates studied. These values reflect the treatment/ fortification effect of substrates on the physical parameters before fermentation. Viscosity, particle size and zeta potential values of SIW are lower than those found in AnaeroD-SIW and AeroD-SIW. Whereas, substrates treated at pH 2 showed lower values of viscosity, particle size and zeta potential than untreated substrates. Substrate treatment at pH 2 leads to the solubilization of organic matter, which resulted in a reduction of viscosity, particle size and zeta potential. When the SIW substrate was fortified with aerobic or anaerobic effluent or SIW solids, the SIW viscosity, particle size, and zeta potential values increased with an increase in the concentration of the added solids.

A substrate's viscosity plays a crucial role in oxygen transfer from the gas phase to the liquid phase. When a medium is highly viscous, it does not provide optimally or required oxygen transfer, and the oxygen transfer coefficient (KLa) measurement shows a low oxygen transfer. As a result, bacterial cell growth, sporulation and endotoxin synthesis are affected [4, 7].

SIW particles showed a tendency to coagulate in solution, whereas aerobic and anaerobic effluent shows colloid stability. Aerobic and anaerobic effluent is rich in microorganisms, mostly negatively charged bacteria and polymeric substances (negatively charged) synthesized by these microorganisms [25, 26]. The high presence of these negatively charged entities in the diffuse layer and the surface of colloids favors repulsion between colloidal particles as indicated by the zeta potential stability, described by the double-layer theory Derjaugin, Landau, Verwey, and Overbeek: DLVO [27].

The zeta potential characteristic in anaerobic and aerobic effluent could stabilize viscosity, avoiding particles' coagulation during fermentation. In [4], it was described that particles' interaction during fermentation would be sufficient to cause high viscosity. Moreover, the stability of zeta potential in anaerobic and aerobic effluent could be advantageous in biopesticide formulation. Particle dispersion in a formulation is an important property required by the addition of additives such as sorbitol [4, 8].

Entomotoxicity bioassay

After centrifugation and formulation, entomotoxicity of the different formulations obtained from the fermented broth of different substrates was measured (Table 6). Entomotoxicity obtained from AeroD-SIW, and AnaeroD-SIW SIW formulations (pre-treated at pH 2) displayed significantly higher than the untreated formulations. The entomotoxicity of SIW treated at pH 2 is even equivalent to the standard Foray 76B. The entomotoxicity value of fortified AeroD-SIW, and AnaeroD-SIW and SIW solids at 15 g/L showed results below or equivalent to those obtained without fortifications. Further, the fortified SIW formulations with AeroD-SIW, and AnaeroD-SIW solids at 30 g/L exhibited lower (AeroD-SIW, and AnaeroD-SIW) or slightly superior (fortification with SIW) entomotoxicity results than obtained from substrates without fortification. In [28], it was indicated that the spores and crystals mixture is 4–5 times more pathogenic than a solution containing either. This explains the high entomotoxicity of larvae in SIW fortified with SIW and SIW fortified with AneroD-SIW as the two medium show high crystals synthesis and spores (Table 6, Fig. 3). The entomotoxicity in SIW formulation treated at pH 2, SIW fortified with SIW and AnaeroD-SIW was greater than Foray 76B (commercial biopesticide), which was 20,000 IU/µL. This value was described as sufficient for the treatment of a surface area of 1 ha by a 1.5 L volume [29]. Thus, these formulation would be sufficient to increase the biopesticide efficiency [17]. The unit production cost for Btk-based biopesticide is 2.54 $/L, which is less than that of chemical pesticides (4–15 $/L) [17].

Table 6 Entomotoxicity of the different formulation prepared from fermented broths of different substrates


These experiments investigated Bacillus thuringiensis var kurstaki (Btk) culture using starch industry wastewater (SIW), anaerobic and aerobic effluent obtained from starch industry wastewater. The results showed that anaerobic and aerobic effluent did not contain enough total organic carbon to augment Btk growth and entomotoxicity. At the same time, substrates pre-treated at pH 2 provided significant organic matter solubilization for Btk culture and resulted in larvicidal potency equivalent to the commercial biopesticide Foray 76B. SIW fortification with 30 g/L concentrated SIW increased the formulation entomotoxicity.

Availability of data and material

All data generated or analyzed during this study are included in this article.

Code availability

Not applicable.


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The research has been funded by Natural Sciences and Engineering Research Council of Canada (Grants A4984, STR 202047, SCF 192190-96 and Canada Research Chair).

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Correspondence to Adama Ndao.

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Ndao, A., Sellamuthu, B., Kumar, L.R. et al. Biopesticide production using Bacillus thuringiensis kurstaki by valorization of starch industry wastewater and effluent from aerobic, anaerobic digestion. Syst Microbiol and Biomanuf 1, 494–504 (2021).

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  • Bacillus thuringiensis
  • Aerobic digested effluent
  • Anaerobic digested effluent
  • Effluent valorization
  • Biopesticide