Research Article
Improvement of stability of insecticidal proteins from Bacillus thuringiensis against UV-irradiation by adsorption on sepiolite
Adsorption Science & Technology 0(0) 1–13 ! The Author(s) 2018 DOI: 10.1177/0263617418759777 journals.sagepub.com/home/adt
Xueyong Zhou, Hang Li, Yanmei Liu, Jianchao Hao, Huifen Liu and Xianzhi Lu Tianjin Agricultural University, PR China
Abstract The adsorption characteristics of protoxin and toxin from Bacillus thuringiensis by sepiolite were studied. The kinetic results showed that the protoxin and toxin could be adsorbed by sepiolite rapidly, and the equilibrium was reached within 1 h. The adsorption isotherms of both proteins followed Langmuir equation (R2>0.98) and the curves belonged to L type. The adsorption capacity, adsorption rate and the insecticidal activity of toxin were higher than those of protoxin. The results of ultraviolet irradiation showed that sepiolite could protect the toxin from ultraviolet irradiation damage, but it accelerated the damage of protoxin. Therefore, the toxin is suitable for preparing long-term formulations of B. thuringiensis using the carrier of sepiolite. The results of Fourier transform infrared, X-ray diffraction, and scanning electron microscopy indicated that toxin did not influence the structure of sepiolite and the adsorption of toxin only on the surface of sepiolite. The adsorption of toxin by sepiolite was enhanced by metal ions in the range of concentration from 0 to 4.0 mmol L1, and the enhancement extent of metal ions on the adsorption of toxin was in the order: Ca2þ>Mg2þ>Kþ>Naþ. The higher the loading of toxin per mass sepiolite, the lower the 50% of the larvae of sepiolite–toxin complex. The zeta potential of sepiolite suspension increased with the addition of metal ions, indicating that the driving forces of the adsorption of toxin to negatively charged sepiolite was electrostatic interaction.
Corresponding author: Xueyong Zhou, College of Food Science and Bioengineering, Tianjin Engineering and Technology Research Center of Agricultural Products Processing, Tianjin Agricultural University, Tianjin, PR China. Email:
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Keywords Bacillus thuringiensis, protoxin, toxin, sepiolite, adsorption, insecticidal activity Submission date: 11 September 2017; Acceptance date: 16 January 2018
Introduction Bacillus thuringiensis (Bt) has been extensively used for five decades in biopesticidal formulations due to its safe environmental and human health records. It is cultured in commercially available liquid media and spray-dried to produce high concentrations of raw materials (technical powder) including mainly crystal proteins and endospores for formulation (Brar et al., 2006; Zhou et al., 2004). The crystalline protein is not toxic and requires solubilization and enzymatic cleavage to yield the active toxin whose molecular weight is 60–70 kDa (Aronson et al., 1991; Herna´ndez and Rodrigo, 2004). The widespread use of Bt is often challenged by production as well as formulation costs. Formulation is a crucial link between production and application, and dictates economy, longer shelf life, ease of application and enhanced field efficacy. There are various environmental factors like ultraviolet radiation, rain, pH, temperature and foliage physiology which impede efficacy of Bt formulations. One disadvantage of Bt formulations is that crystalline protein and spores are rapidly inactivated by sunlight or degraded by microorganisms when they are applied in the fields (Brar et al., 2006; Koskela and Stotzky, 1997; Liu et al., 1993; Pozsgay et al., 1987). Hence, how to prolong the residual period of B. thuringiensis formulations has received extensive attention. To accomplish this, commercial preparations of B. thuringiensis are mixed with anti-ultraviolet absorbents, encapsulated in a coating (capsule) made of gelatin, starch, cellulose and other polymers and even encapsulated within microbial cells (Brar et al., 2006). An economically, efficient method for the preparation of long-term formulation of B. thuringiensis, however, is still indispensable. Previous studies indicated that the protoxin and toxin proteins from B. thuringiensis, could be sorbed rapidly on clay minerals, humic acids and organo-mineral complexes (Helassa et al., 2009; Muchaonyerwa et al., 2006; Venkateswerlu and Stotzky, 1992; Zhou et al., 2005), and their insecticidal activity was comparable with or higher than that of the free proteins (Crecchio and Stotzky, 1998, 2001; Saxena et al., 2010). The adsorption or immobilization of insecticidal proteins on clay minerals may provide a new approach for the preparation of long-term formulations of B. thuringiensis, however, available data showed that the loading capacity of toxin on minerals was low. The adsorption of toxin tended to equilibrium at a toxin/ adsorbent ratio of 5:1 and the amounts of adsorption on kaolinite, goethite and silica were 0.45, 0.43, and 0.25 g g1, respectively, and the corresponding percent adsorptions of toxin were 10.0%, 9.5% and 5.5% (Zhou et al., 2005). Therefore, some new measurements for improving the adsorption efficacy of Bt toxin onto clay minerals deserves further studies. Sepiolite is commercially obtainable, of relatively low cost and non-toxic. Main deposits of sepiolite are located in Anatolia in Turkey, Ceelbuur in Somalia, South-central China and Spain with 70% of world reserves and annual output of approximately 1,300,000 tons (Hrenovic et al., 2010). There is an increasing interest in recent years to utilize sepiolite in environmental studies since sepiolite is a good adsorbent of neutral molecules and cations
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(Alkan et al., 2005). Sepiolite particles are needle-like and does not exhibit swelling properties (Can et al., 2010), however, it has a high specific surface area (SSA, 300 m2 g1), that allows adsorption of pesticides (Gonza´lez-Pradas et al., 2005; Maqueda et al., 2009). Up to now, we are not aware of any studies regarding the adsorption characteristics of sepiolite for the insecticidal proteins from B. thuringiensis. The present study was aimed to evaluate the adsorption potential of sepiolite for two contrasting Bt proteins: protoxin (130 kDa) and toxin (65 kDa), and the insecticidal activity of Bt proteins before and after adsorption was determined. On this basis, the enhancement of metal cations for the loading of toxin was studied. These results will give important reference to the development of adsorption formulation of B. thuringiensis.
Materials and methods Sepiolite The sepiolite, a commercial sample from Nanyang Leibao Sepiolite Processing Company Limited in China was dry-sieved under open laboratory conditions using the British Standard (300 standard mesh sieve) and stored in a polypropylene container. The mineral characterization (Table 1) was performed determining its SSA by the single point BET N2 adsorption method, its cation exchange capacity (CEC) by the ammonium acetate method (Tan, 1996) and its mineralogical composition was identified by X-ray diffraction (XRD). The zeta potential of sepiolite was determined by electrophoretic mobility measurements on a Nano-ZS90 ZETASIZER (Malvern Instruments, UK). The zeta potential was plotted versus pH values, and the isoelectric point (IEP) was obtained according to the intersection point between the curve and horizontal coordinate.
Preparation of protoxin and toxin A powder of B. thuringiensis subsp. kurstaki (the type of crystal protein is Cry1Ac) was provided by the State Key Laboratory of Agricultural University. Four gram of the powder was washed three times with 0.5 mol L1 NaCl and three times with double distilled water (ddH2O). Crystals and spores were removed from suspension liquor by centrifugation (15,000 g for 15 min) and resuspended in 0.05 mol L1 NaOH þ 0.1 mmol L1 PMSF (phenylmethylsulfony1 fluoride) for 15 min. The aqueous suspension was centrifuged at 20,000 g for 15 min (4 C) and insoluble materials were discarded. The supernatant was transferred into, 1000 mL beaker, and the solution was titrated by 25% (v/v) acetic acid under agitating until the pH was near 4.4. The suspension was agitated for 15 min and the pH was verified again. The suspension was allowed to stand for 4 hours then part of supernatant was carefully removed by siphon pipe. The protoxin precipitate was obtained by centrifugation at 20,000 g for 10 min and then lyophilized. The molecular weight of the protoxin was 130 kDa determined by polyacrylamide gel electrophoresis Table 1. Mineral characteristics of sepiolite. CEC (cmol kg1)
SSA (m2 g1)
Isoelectric point
Zeta potential (mV)
4.0
141
6.6
55
CEC: cation exchange capacity; SSA: specific surface area.
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(PAGE) and no degradation of protoxin was observed during the purification. The active toxin was prepared from Bt powder (containing crystals and spores) using 0.05 mol L1 sodium hydroxide. Different from the preparation of protoxin, Bt powder was only washed two times with double distilled water (ddH2O), then the precipitate was resuspended in 0.05 mol L1 NaOH solution for 60 min in 37 C water bath. The residual protease is enough to break protoxin into toxin. The pH of the supernatant liquor was adjusted to 4.4 as described above. The toxin precipitate was centrifuged at 20,000 g and then lyophilized. The molecular weight of the protein was about 65 kDa as determined by PAGE.
Adsorption of the protoxin and toxin The protoxin and toxin were dissolved in Tris buffer (pH 8.0). The insoluble material was discarded after centrifugation at 20,000 g for 15 min. The sepiolite powder was suspended in sterilized deionised water at a concentration of 10 g L1. Procedures used for determining the concentrations of insecticidal proteins by means of optical density measurements at 280 nm (A280) using pure protoxin or toxin from B. thuringiensis as standard. The adsorption was investigated at different time intervals between 0.5 and 5 h. The adsorption isotherms were measured for initial protein concentrations from 0.25 to 1.0 g L1 and adsorbent content of 1.0 g L1 at pH 8.0. The mixtures were shaken at 25 1 C for 3 h. The dispersions were centrifuged at 20,000 g for 20 min and the absorbance of supernatants was measured. The amount of protein adsorbed (g g1) was calculated from the concentration difference. Control experiments were performed with an adsorbent in the absence of a protein. The effect of metal ionic (Naþ, Kþ, Ca2þ and Mg2þ) on the adsorption of toxin was measured in the range of concentration from 0 to 4.0 mmol L1 at a toxin concentration was 0.5 g L1 and a sepiolite concentration of 1.0 g L1.
XRD analyses After adsorption, the sepiolite–protein complex was lyophilized, and then the sample was identified by XRD analysis (JDX-10P3A, Japan).
Determination of insecticidal activities The insecticidal proteins adsorbed on sepiolite was diluted to 100 lg mL1. Twenty milliliters of the diluted solution was transferred into a Petri dish (diameter 10 cm), which was placed under an ultraviolet lamp. The power of ultraviolet lamp was 20 W, and wave length was 253.7 nm. The vertical distance between the lamp and the Petri dish was 30 cm. The Petri dish was shaken for 10 s every 15 min. After one hour’s irradiation of ultraviolet, the insecticidal activity of proteins adsorbed on sepiolite was determined. Heliothis armigera was used to determine the insecticidal activity of these proteins. Free and adsorbed proteins were diluted to 200, 100, 50, 25, 12.5 and 6.25 lg mL1. The inflection feed was prepared as follows: agar powder (4.5 g) was dispersed in 220 mL sterilised water, and maintained boiling for 10 min. Yeast powder (12 g), soybean powder (24 g), sodium benzoate (0.42 g) and vitamin C (1.5 g) were mixed and soaked with 80 mL sterilized water, then transferred into the agar solution. Under stirring, the temperature of the suspension solution dropped to 60 C, and 3.9 mL acetic acid (36%, w/w) was added. The inflection feed was obtained by mixing 4 mL of insecticidal sample with 16 mL suspension feed. Twenty
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milliliters of inflection feed was dispersed into 24 wells of an incubation plate. After airdrying for 12 h, one larva was placed into each well and incubated at 30 2 C for 72 h. Forty-eight larvae were used for each concentration of insecticidal protein (two incubation plates). Mortality was then determined and the lethal concentration to kill 50% of the larvae (LC50) was calculated. The powder of B. thuringiensis and sterilized sepiolite were used as controls.
Infrared spectroscopy and scanning electron microscope Fourier transform infrared (FTIR) adsorption spectra of sepiolite before and after adsorption of Bt toxin were recorded on a Nicolet AVATAR 330 spectrometer using KBr pellets. The surface morphology of sepiolite before and after adsorption of Bt toxin was examined on a Hitachi S-4800 scanning electron microscope (equipped with an X-ray dispersive analyzer).
Statistics Data are expressed as average the standard deviation. Unless indicated otherwise, the standard errors of the means are within the dimensions of the figure symbols.
Results and discussion Effects of molecular weight of Bt proteins on adsorption kinetics The adsorption of protoxin or toxin by sepiolite increased rapidly, and the equilibrium was reached within 1 h (Figure 1). Before equilibrium, the adsorption rate of toxin was higher than that of protoxin. After 3 hour’s contact, the adsorption of the protoxin and toxin was 0.16 and 0.22 g g1, respectively. No remarkable increase in adsorption was observed after 3 h. Therefore, a contact time of 3 h was chosen to prepare the sepiolite–protein complex.
Figure 1. Effect of contact time on adsorption of protoxin and toxin.
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Table 2. Langmuir parameters for adsorption of protoxin and toxin. Langmuir parameters Insecticidal proteins
KL (mL mg1)
Umax (mg mg1)
R2
Toxin Protoxin
0.7178 1.031
1.140 0.7348
0.9949 0.9884
Figure 2. Isotherms of adsorption of protoxin and toxin on sepiolite.
Effects of molecular weight of Bt proteins on adsorption isotherms The adsorption data were fitted to the Langmuir equations with high regression coefficients (Table 2). The Langmuir equation used was the Hanes–Woolf linearized form (Vettori et al., 1999; Zhou et al., 2005): C/C=C/Cmaxþ1/Cmax KL, where C is the concentration of protein in solution at equilibrium (g L1), C is the amount adsorbed (g g1 of mineral), Cmax is the maximum adsorption capacity of the clay (g g1 of mineral), and KL is the Langmuir constant (L g1). A plot of C/C vs. C yields an intercept on the ordinate is 1/Cmax KL and a slope 1/Cmax. In the range of protein concentrations from 0.25 to 1.0 g L1, the adsorption amounts increased rapidly at the initial stage and then reached equilibrium gradually (Figure 2). The equilibrium adsorption isotherms of the insecticidal proteins on sepiolite were of the L type (Giles et al., 1960). The adsorption capacity of toxin was higher than that of protoxin (Table 2). The maximum adsorption of protoxin and toxin was 0.7348 and 1.140 g g1, respectively.
Effects of molecular weight of Bt proteins on the insecticidal activity As shown in Table 3, both free and adsorbed Bt proteins were toxic to the larval of H. armigera. The LC50 values for the free and adsorbed toxin was 10.0 and 3.32 lg mL1, for the free and adsorbed protoxin was 14.0 and 4.85 lg mL1. The LC50 of adsorbed Bt
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Table 3. Insecticidal activity of Bt proteins before and after adsorption by sepiolite. Samples
LC50a lg mL1
LC50 after UV lg mL1
Percent changing of LC50 after UVb %
Protoxin Toxin Protoxin-sepiolite Toxin-sepiolite Powder of B. thuringiensis
14.0 10.0 4.85 3.32 28.4
19.8 13.1 7.83 3.66 36.2
41.4 31.0 61.4 10.2 27.5
Bt: Bacillus thuringiensis; LC50: 50% of the larvae. a Lethal concentration to kill 50% of the larvae. b Change ¼ [(LC50 after ultraviolet irradiation LC50 before ultraviolet irradiation)/LC50 before ultraviolet irradiation] 100.
proteins were lower than those of free proteins, which was in agreement with a previous study (Crecchio and Stotzky, 2001). The higher insecticidal activity of bound protein may have been a result of the protein being concentrated by binding on the complexes, and consequently, more protein was ingested by the larvae than the free protein was uniformly spread over the surfaces of food medium (Crecchio and Stotzky, 1998). The ability of bound protein to kill larvae may have been a result of the higher affinity of receptors on the epithelium of the larvae gut for the protein than the lower affinity, because of nonspecific binding of protein for the mineral complexes. The movement of the protein from the minerals to specific binding receptors was probably a result of the fact that the binding of protein on minerals is primarily by H-bonds, and the segments of bound proteins are constantly detaching and reattaching. After one hour of ultraviolet irradiation, the LC50 of the free protoxin and toxin increased by 41.4% and 31.0%, respectively, whereas the adsorbed protoxin and toxin increased by 61.4% and 10.2%, respectively. The results showed that sepiolite could protect the toxin from ultraviolet irradiation damage, but accelerated the damage of protoxin by ultraviolet irradiation. The differences in ultraviolet degradation between protoxin and toxin may be due to differences in their molecular weight. Protein is a kind of chain molecules, and the larger the molecular weight, the longer the molecular chain. As the molecular weight of protoxin was two times larger than that of toxin, the molecular chain of protoxin is far longer than that of toxin. The free protoxin with a long chain will be automatically folding, which protects the inner part from the ultraviolet irradiation damage. When the protoxin is adsorbed by sepiolite, its molecules will be stretched and fixed on the mineral surface, which is easily damaged by the ultraviolet irradiation. The present study indicated that both adsorption amount and insecticidal activity of toxin were higher than those of protoxin, therefore, the toxin is suitable for preparing long-term formulations of B. thuringiensis using the carrier of sepiolite.
XRD analysis of sepiolite before and after adsorption of toxin The XRD pattern of sepiolite before and after adsorption of toxin is shown in Figure 3. The d001 of sepiolite, sepiolite–toxin and sepiolite-protoxin was 1.217, 1.213 and 1.217 nm,
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Figure 3. XRD analysis of sepiolite before and after adsorption of Bt proteins. XRD: X-ray diffraction; Bt: Bacillus thuringiensis.
Figure 4. FTIR analysis of sepiolite–toxin complexes. FTIR: Fourier transform infrared.
respectively. The binding of toxin almost did not cause expansion of sepiolite. It was suggested that the toxin did not intercalate sepiolite, as the amounts of expansion were too small. Therefore, the toxin was only adsorbed on the surface of sepiolite.
FTIR analysis of sepiolite–toxin complexes As shown in Figure 4, the spectrum of the sepiolite showed the band at, 3687 cm1 corresponding to Mg–OH vibration inside the sepiolite block and the band at 3560 cm1 assigned to the vibrations of water coordinated to the Mg2þ ions located at the edges of the structural
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Figure 5. SEM Analysis of sepiolite–toxin complexes. SEM: scanning electron microscopy.
Figure 6. Effects of metal ions on adsorption of Bt toxin by sepiolite. Bt: Bacillus thuringiensis.
blocks (Eren and Gumus, 2011; Song et al., 1998) (Figure 4). Bands at 3435 and 1649 cm1 are assigned to vibrations of water (Maqueda et al., 2009). After the adsorption of Bt toxin (protoxin is similar to toxin, data not shown), methyl, methylene and amide I bands of the complexes occur at 2964, 2929 and 1651 cm1, respectively. The bands at 3686 cm1 corresponding to Mg–OH vibration inside the sepiolite block and the bands at 3560 cm1 assigned to the coordinate water molecules (mOH) (Figure 4) remained unaffected, indicating an absence of water bridges between Mg2þ and the toxin inside the tunnels. Therefore, the FTIR spectra clearly revealed that Bt toxin molecules were not present inside the tunnels, which was coincident to the XRD results.
SEM analysis of sepiolite–toxin complexes Morphology of sepiolite (Figure 5(a)) and sepiolite-Bt toxin (Figure 5(b)) was observed on a scanning electron microscope (SEM, Hitachi S-4800). Before the adsorption of toxin,
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Figure 7. Effects of metal ions on the zeta potential of sepiolite suspension.
natural sepiolite is in the state of aggregation of the typical sepiolite fibers. After the adsorption of toxin, the aggregation degree of sepiolite increased; this may be due to the package action of macromolecular protein to mineral particles.
Effects of metal ions on the loading of Bt toxin by sepiolite When the sepiolite–toxin suspension was adjusted by NaCl and KCl from 0 to 4.0 mmol L1, the adsorption of toxin by sepiolite increased by 26.5% and 48.4%, respectively. When the sepiolite–toxin suspension was adjusted by MgCl2 and CaCl2, an initial steep rise of toxin adsorption was observed until the metal ionic concentration reached 0.5 mmol L1, then adsorption growth tended to slow (Figure 6). At the final metal ionic concentration of 4.0 mmol L1, the adsorption amount of toxin increased by 1.4 and 2.0 times, respectively. The enhancement extent of metal ions on the adsorption of toxin was in the order: Ca2þ>Mg2þ>Kþ>Naþ. Further study indicated that the toxin began to flocculate if the concentration of MgCl2 or CaCl2 was more than 4.0 mmol L1, therefore, the addition concentration of metal ions should be less than 4.0 mmol L1.
Effects of metal ions on the zeta potential of sepiolite The zeta potential of sepiolite suspensions is shown in Figure 7. In the range of metal ionic concentration from 0 to 4.0 mmol L1, zeta potential of sepiolite suspension increased rapidly at first with the increase of metal ionic concentration, then the zeta potential tended to gradually come to an equilibrium. The extent of influence of metal ions on the zeta potential is in the order: Ca2þ > Mg2þ > Kþ > Naþ, which has the same trends as the influence of metal ions on the toxin adsorption. Sander et al. (2010) indicated that electrostatic interactions govern the adsorption of Bt protein by silica. The IEP of sepiolite was 6.6 (Table 1), the IEP of toxin from B. thuringiensis was 5.5 (Keeton and Bulla, 1997). As the experimental pH (8.0) is more than their IEP, the sepiolite and toxin are negatively charged, therefore, there is a repulsive force between mineral and toxin. With the increase of metal ionic concentration, the zeta potential of sepiolite increased (Figure 7),
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Table 4. Effects of metal ions on the insecticidal activity of sepiolite–toxin complex. Metal ions
Ionic concentration (mmol L1)
Toxin adsorbed (mg mg1)
LC50 of sepiolite–toxin complex (lg complex/mL)
Control Naþ Kþ Mg2þ Ca2þ
0 1.0 1.0 1.0 1.0
0.1237 0.1359 0.1628 0.1872 0.2097
30.16 27.45 22.92 19.93 17.79
LC50: 50% of the larvae.
resulting in a reduction of repulsive force between toxin and mineral. The decrease of repulsive force would promote the adsorption of toxin, which agrees with the earlier studies (Liu et al., 2012). Besides, cation such as Ca2þ can play a role as a bridge between carboxyl groups of protein and soil minerals to enhance the adsorption (Fu et al., 2012; Norde 1984). In contrast, Madliger et al. (2010) found that Cry1Ab protein adsorption to SiO2 increased with decreasing I (ionic strength) from I =50 mM to I ¼ 10 mM, and no adsorption of Cry1Ab protein to SiO2 was detected at I ¼ 250 mM. Madliger et al. (2010) used the patch controlled electrostatic attraction (PCEA) to explain their results. They proposed that Cry1Ab protein was adsorbed oriented with the positively charged domains II and III toward SiO2 despite negative net charges on both Cry1Ab and SiO2. In our studies, we indicated that the driving forces of the adsorption of toxin to negatively charged sepiolite was electrostatic interaction, which agreed with the observations obtained from Sander et al. (2010). Further studies are also required to elucidate the interaction mechanism between toxin and minerals.
Effects of metal ions on the insecticidal activity of sepiolite–toxin complex The adsorption of toxin by sepiolite increased with the addition of metal ions in the range of 0–4.0 mmol L1. When the ionic strength was adjusted by Naþ, Kþ, Mg2þ and Ca2þ to 1.0 mmol L1, the insecticidal activity of toxin–sepiolite complex was 30.16, 27.45, 19.93 and 17.79 lg complex mL1 (Table 4), i.e. its LC50 decreased by 8.98%, 24.0%, 33.9% and 41.0%. Obviously, the insecticidal activity of sepiolite–toxin complex increased after the adsorption of toxin was enhanced by metal ions.
Conclusions The protoxin and toxin from B. thuringiensis could be adsorbed by sepiolite rapidly, and the equilibrium was reached within 1 h. The adsorption isotherms of both proteins followed Langmuir equation (R2>0.98) and the curves belonged to L type. The adsorption capacity, adsorption rate and the insecticidal activity of toxin were higher than those of protoxin. The results of ultraviolet irradiation showed that sepiolite could protect the toxin from ultraviolet irradiation damage, but it accelerated the damage of protoxin. Therefore, the toxin is suitable for preparing long-term formulations of B. thuringiensis using the carrier of sepiolite. The results of FTIR, XRD, and SEM indicated that toxin did not influence the structure of sepiolite and the adsorption of toxin only on the surface of sepiolite. The adsorption of
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toxin by sepiolite was enhanced by metal ions in the range of concentration from 0 to 4.0 mmol L1, and the enhancement extent of metal ions on the adsorption of toxin was in the order: Ca2þ>Mg2þ>Kþ>Naþ. The higher the loading of toxin per mass sepiolite, the lower the LC50 of sepiolite–toxin complex. The zeta potential of sepiolite suspension increased with the addition of metal ions, indicating that the driving forces of the adsorption of toxin to negatively charged sepiolite was electrostatic interaction. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research was funded by the National Natural Science Foundation of China (Nos. 31470573 and 31070478).
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