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Optimization of spray drying process for Bacillus thuringiensis fermented wastewater and wastewater sludge
´ • K. D. Vu • R. D. Tyagi • K. D. Adjalle ´ ro • R. Y. Surampalli S. K. Brar • J. R. Vale
Received: 15 April 2010 / Accepted: 26 August 2010 / Published online: 11 September 2010 Ó Springer-Verlag 2010
Abstract Response surface methodology was used to optimize spray drying process for producing biopesticide powders of Bacillus thuringiensis by using fermented broth of starch industry wastewater and wastewater sludge. Analysis of variance was carried out using number of viable spores in the powder as dependent variable. The determination coefﬁcients of models were 92 and 94% for fermented broth of starch industry wastewater and wastewater sludge, respectively. Under the optimal conditions of the operational parameters of spray drying, the numbers of viable spores were 2.2 9 108 and 1.3 9 108 CFU/mg in the dry powders for starch industry wastewater and wastewater sludge respectively, with a loss of viable spores of 18 and 13% when compared with their respective fermented broths. The entomotoxicity (measured by the bioassay method) of the powders obtained under optimal conditions showed a loss of 28 and 18% when compared with the fermented broth of starch industry wastewater and wastewater sludge, respectively. The optimized results of spray drying were used for ﬁeld application calculations. The volume of fermented broth required to produce powder formulated product when compared with the volume
required for liquid formulation product in order to treat 1 ha of balsam ﬁr was less and offered several advantages. Keywords Bacillus thuringiensis Á Wastewater Á Sludge Á Spray drying Á Response surface methodology Abbreviations ANOVA Analysis of variance Bt Bacillus thuringiensis CCD Central composite design CFU Colony forming units IU International units R2 Coefﬁcient of determination RSM Response surface methodology SBU Spruce budworm units SIW Starch industry wastewater T Temperature TH Thermo-hydrolyzed sludge Tx Entomotoxicity VS Number of viable spores count
Introduction Biopesticides derived from bacteria, fungi, viruses and protozoa are gaining wide interest due to the adverse impacts of chemical pesticides. Among the biopesticides, Bacillus thuringiensis (Bt), var. kurstaki, is a widely employed biocontrol agent [1, 2]. Final product formulations of Bt comprise mixture of spores and insecticidal crystal proteins obtained after growth and sporulation in a bioreactor and settling of fermented broth. The biopesticides of Bt are usually marketed as solid and liquid formulations.
´ Á K. D. Vu Á R. D. Tyagi (&) Á K. D. Adjalle ´ ro S. K. Brar Á J. R. Vale ´ du Que ´ bec, INRS-ETE, Universite 490, Rue de la Couronne, Quebec, QC G1K 9A9, Canada e-mail: email@example.com R. Y. Surampalli US EPA, P.O. Box 17-2141, Kansas City, KS 66117, USA
(3) estimation of the volume of fermented broth required to produce powder formulation product in order to treat 1 ha of balsam ﬁr when compared with the volume required for liquid formulation product. whereas secondary sludges were pre-treated (hydrolyzed) at 140 ± 1 °C. At the entry of nozzle. The drying parameters which affect the viability of the cells and spores include air inlet and outlet temperatures. wettable powders obtained by drying the fermented broths containing the active components (spores. Spray drying process Yamato spray dryer. and insecticidal crystal proteins) depend on the suitable operational parameters. However. more than 90% of the moisture is evaporated in the ‘‘drying chamber’’ (Yamato manual). In this study. of Yamato Scientiﬁc America. The fermented broth sample was conveyed to the spray dryer nozzle by means of a peristaltic pump. culture medium and fermentation The bacterial strain used in this study was Bacillus thuringiensis var. 1. 7]. The principle of operation is detailed in a process scheme in Fig. Materials and methods Bacterial strain. it would be interesting to evaluate the possibility and feasibility of the production of powders from Bacillus thuringiensis fermented broths obtained with these two media. QC. the spray drying process seems to be appropriate [6. 123 . the fermented starch industry wastewater and hydrolyzed wastewater sludge will be referred to as SIW and TH. Quebec) with bioreactor of 15 l (Bioge accessories. 5]. Drying is regarded as the suitable method of conservation to prevent contamination by micro-organisms [4. Fermentation was conducted in a ´ nie Inc. QC. namely. temperature. The originality of this work lies in the fact that the fermented broth of Btk used for the spray drying process was fermented using wastewater and wastewater sludge that poses speciﬁc problems due to the complex nature of the substrates. the dried powder particles were collected in the ‘‘product vessel’’ after separation from the moisture vapor through the ‘‘cyclone’’. Canada). particles sizes. the solid formulations have the advantages of longer shelf-life. respectively. the conditions of drying affect the viability of the spores and the efﬁcacy of the insecticidal crystal protein.5 and the fermented broth was aseptically collected in a sterilized container for drying by spray dryer. and ease of transport .238 Bioprocess Biosyst Eng (2011) 34:237–246 Compared with the liquid formulations. The diameter of liquid droplets obtained was approximately 20 lm with a surface area of 3. If the surface of contact of the droplets and the hot air is large. which require optimization. enzymes) are widely used. hot air ﬂow rate and feed rate) of fermented broths of these two media by using the central composite design (CCD) and response surface methodology (RSM). Initial studies carried out in our laboratory on development of liquid formulations from fermented wastewater and wastewater sludge have yielded encouraging results  leading to dry powder formulations. at 40 psig pressure for 30 min. For these reasons. 12]. Among the solid formulations. among others). kurstaki (Btk) HD-1 (ATCC 33679). the drying process affects the size of the particles which is an important parameter in the efﬁcacy of action during ﬁeld application of the product [11. spores. agitation and anti-foam. . at a pressure of 15 psig for 30 min. The detailed procedure of fermentation has been already described by Brar et al. (2) evaluation of the impacts of these viable parameters on spores and entomotoxicity of Bt and. Model Pulvis GB 22.711 mm for SIW and 1 mm for TH) were used. Similarly. It is important to note that the spray dryer was periodically cleaned between runs. Henceforth. spraying pressure. the pH was lowered from 7 to 4. Among the industrial methods of production of wet powder by drying.5. insecticidal crystal proteins. the objectives of the present study were: (1) determination of optimal condition of drying (values of inlet temperature. Looking at the importance of dry formulations. The media used for spray drying in this study were the fermented broths of Btk obtained using different substrates. type of atomization and/or pulverization and the strain of organism [7–10]. enzymes. insecticidal crystal proteins. (1) secondary sludge from wastewater treatment ´ Urbaine de Que ´ bec. and taking into account the rheology of starch industry wastewater and wastewater sludge. it is important to optimize the operational drying parameters to limit the negative impacts of drying on the active components of Bt (spores. two different nozzles (0. Ste Foy and. and the viability and potential of active components (cells. CA. Finally. (2) plant of Communaute starch industry wastewater from ADM-Ogilvie (Candiac. intellution software. sample was mixed with air under pressure (controlled by a needle valve) and the mixture was spray dried in a ‘‘drying chamber’’. USA) for the control of pH. The characteristics of the powders (moisture. due to the large difference of concentrations between the two fermented broths. At the end of fermentation. The droplets were instantaneously dried by hot air in the ‘‘drying chamber’’. solubility) obtained. connected to a computer with the iFix 3. Inc (South San Francisco. USA) was used for the drying of different fermented broths. air ﬂow. The work volume in the bioreactor was 10 l. (MA. Thus. Starch industry wastewater was directly used for fermentation after in situ sterilization at 121 ± 1 °C.00 cm2 per ml of the sample (as per Yamato instructions manual)..
125 g of TH powder in 5 mL sterilized water was used. values of linear coefﬁcients. the mixture prepared possessed the same concentration as the fermented broths of SIW and TH. between these drying operational parameters. The results of the CCD experiments and measurement of the dependent variables (moisture content. viable spores count (VS) and entomotoxicity (Tx) are dependent variables. (2) rate of feed which deﬁnes the concentration of the sample and determines the water content in the air–sample mixture. inlet air temperature (X3) and the pressure of atomization of air (X4) are regarded as independent variables. to evaluate the interactions if there is any. The effects of independent variables on the Y response of the dependent variables were analyzed according to a polynomial model of second order of surface response given by the following general equation: n n X X Y ¼ Ao þ Ai Xi þ Bij Xi Xj ð 1Þ i ¼1 j i Fig. Ai. ON. on one hand. it was possible to determine the optimal values of these parameters. and (4) ﬂow rate of aspiration of hot air which determines the duration of contact between the hot air and droplets of pulverized sample–air mixture. Experimental design Independent and dependent variables In the statistical response surface method. viable spores. These parameters will make it possible to obtain low moisture content. a suspension containing 0. By applying the RSM. number of viable spores and entomotoxicity) will be analyzed by STATISTICA 6 of STATSOFT Inc. intercept. entomotoxicity.075 g of SIW and 0. predicted response. to precisely evaluate the signiﬁcant effects of various parameters on the moisture content. Thus.85% w/v) before the counting of viable spores which was carried out according to the procedure described by 123 . 1 Schematic of spray dryer The important parameters of spray dryer are as follows: (1) inlet temperature which measures the degree of hot air. this statistical RSM will make it possible. Measurement of dependent variables Outlet temperature and moisture content The outlet temperature is measured directly at the exit port of the powders by the corresponding probe (Fig. the operational parameters. Canada). The water content (moisture) of different dry powders was obtained by drying the samples by using HR 83 Halogen Moisture analyzer (Mettler 120 Toledo. 1). ﬂow rate of hot air (X2). Additionally. Viable spores and entomotoxicity For the counting of viable spores and the entomotoxicity measurements. and on the other hand. Central composite design and response surface methodology In continuation of the experiments to determine the provisional optimal values of the independent variables. where Y. (Thulsa.Bioprocess Biosyst Eng (2011) 34:237–246 239 Method of ascending slope The method of the ascending slope makes it possible to follow the direction of the response while varying the independent variables . Ao. such as feed rate of the pump (X1). a central composite plan will be drawn to cover the relevant values of the independent variables. moisture content (M). crystal proteins and entomotoxicity. (3) pressure of atomization of air deﬁning the size and surface of the droplets. high concentrations of viable spores. values of various levels of the independent variables. The outlet air temperature (Toutlet) (which can be controlled by inlet temperature and feed rate). US) by surface response methodology. Xi and Xj. The orientation of the slope follows the direction so that it increases while the dependent variable is decreased and the independent variables are increased. values of quadratic coefﬁcients. A study of determination of the provisional optimal values of these variables was carried out by using the steepest ascent method. The mixtures so obtained were diluted in sterilized saline water (0. and Bij. and covering a large interval of the parameters as described above. outlet temperature.
as well as results of the dependent variables [outlet temperature (Toutlet). For the two media. SIW and TH with 30 ml of diet.49 0.50 7 0.61 140 150 160 170 180 190 200 210 140 150 160 170 180 190 200 210 0.140 0.55 0.155 0. and X4 were 0.33 1 0.095 0. a larva was introduced into each tube.1 1.2 7.10E?10 3.30E?09 3. The tubes were capped by perforated caps and kept at 25 ± 1 °C for 7 days.2 1.49 0.65E?10 8.080 0. Once the diet solidiﬁed and cooled.8 1. Buchler instrument. In the case of TH. the values of moisture content were lower than 11% (w/w). 2) allotted to the four independent variables. ON. respectively with response for viable spores of 1.58 0.20E?10 4.110 0.4 1. 0.140 0.095 MPa. It comprised heating of the sample to 80 °C for 10 min in an oil bath (Thermo-lift.2% (w/w).050 0. spray drying removes more than 90% (w/w) of moisture . USA) followed by rapid cooling in a cold water bath before spreading on the tryptic soya agar plates. The solids concentration affected the ﬁnal spray drying process which has been discussed in details later. Tables 3 and 4 present the experimental plan with 7 replicates at the center. respectively. Adjalle About 0.050 0. X2.80E?09 5.080 0.9 1.75E?10 4.45 5 0.40 0.4 1.75 ml of each dilution was mixed with 15 ml of diet comprising different diet components . X3. ﬁve dilutions were performed in saline water and the last three dilutions were used for the entomotoxicty measurements as described by ´ et al. Thus.48 6 0. 180 °C and 0. 0.90E?09 3. 0. Consequently. These values will make it possible to establish the CCD plan of the experiments. X3 inlet air temperature and X4 pressure of atomization of air 123 .4 9.45E?09 9.80E?10 7.32 8 0.90 9 107 CFU/mg) with a moisture content of 4. The standard deviation of counting was 7–8%. 1.38 2 0.46 0. Table 2 presents the principal values at ﬁve levels (-2.23 2 0.240 Bioprocess Biosyst Eng (2011) 34:237–246 Vidyarthi et al. The mixture thus obtained was distributed in a stack of 10 glass tubes of dimensions 15 mm 9 45 mm (VWR. and the provisional optimal values of the independent variables.40 0.8 8. X2 ﬂow rate of hot air. Steepest ascent method The results of the experiments of the method of the steepest ascent for the two media are presented in Table 1.6 1.8 1. In the light of these results. following analysis and interpretations were drawn: Table 1 Results of the steepest ascent method Results and discussion Composition of the medium and concentration of fermented broths The solids concentration of the fermented broths of starch industry wastewater and wastewater sludge used to feed the spray dryer were 15 and 25 g/l. the high values of viable spores will make it possible to determine the provisional optimal values for the CCD.61 0.52 0. X1. Other controls were prepared by mixing 1 ml of sterilized medium of soya.55 0.43 0.43 0.2 3. Data were subjected to ANOVA for determination of signiﬁcant differences. For the two media.90E?10 3. For SIW.65E?10 5. -1. showed that experiment no 5 (Table 1) gave a high value of viable spores (3.45 g/min.5 8. When optimized.43 4 0.58 0. for the given suspensions of 5 ml prepared with powder of SIW and TH.24 3 0.52 0.125 0.40 9 107 CFU/mg.53 8 0.46 0.110 0.20E?10 3. mortality was evaluated in each stack of 10 tubes. except for the central point where there can be replications.30 7 0. .9 1.27 5 0. Each of the 30 tubes containing 1 ml of diet was used as a control.125 0. X3. the count of viable spores was done.90E?09 7.52 m3/min.155 36 38 39 43 55 73 75 80 36 38 39 43 55 73 75 80 10. moisture content (M). 170 °C and 0. the condition that made it possible to determine the optimal provisional values was given by experiment no 4 (Table 1).55 0.26 4 0.29 g/min.9 8. Provisional optimal values of the independent variables.065 0. X1. The entomotoxicity measurements were carried out by using 2nd instars spruce budworm larvae (Choristoneura fumiferana). Canlab.40 3 0.065 0. X2.59 m3/min.5 8. Central composite design and response surface methodology The CCD consists of an experiment. Mississauga. respectively.60E?09 8. the experimental plan and the results n0 X2 X3 (°C) X4 Toutlet M X1 (g/min) (m3/min) (MPa) (%) VS (CFU/g) Starch industry wastewater 1 0.11 MPa. based on the method of Beegle . for 24 h.29 6 0.06E?10 Secondary hydrolyzed sludge X1 feed rate of the pump. Canada). the optimal values of the independent variables should make it possible to obtain a moisture lower than 11% (w/w) for the biopesticide powders. number of viable spores (VS) and entomotoxicity (TX)]. In fact. The plates were incubated at 30 °C. .40E?10 4. and after.095 0. and X4 were 0.
47 170 0.11 9 108 SBU/g).095 0.55 0. X3 = 180 °C and X4 = 0. airﬂow rate) which affect the viability of the spores and thus decreased the viable spores count. respectively. This high value of moisture content for experiment no 18 (13.075 0. number of viable spores count in the SIW medium.35 g/min).50 0.39 160 0.32 0. feed rate. ANOVA for the two media demon2 2 2 strated that the quadratic effects (X2 1. X4) of the four independent variables were all negative and signiﬁcant. The coefﬁcient of determination. Thus. hot air rate (X2).59 180 0. 1 by ANOVA method showed that the regression model resulted in coefﬁcients of determination.0002). X2. X3.110 0.42 160 0. X3 produced statistically signiﬁcant positive effects (P \ 0. X1 = 0.2 9 108 CFU/mg as compared to that of 2. 2a. This is explained by the combined negative impact of the independent variables (inlet and outlet temperatures.085 0. In this study. These outlet temperatures were slightly lower than those obtained by Xueyong et al. . This optimal condition gave a spore count of 2. Viable spores An analysis of the variance is carried out to determine the ‘‘lack of ﬁt’’ and the signiﬁcant effect of the independent variables on the viable spores (VS) of the two media (SIW and TH). It is important that this percentage is at least higher than 80%. while being positive. This experiment also gave lower value of entomotoxicity (1. and were far from largely affecting the spores and the insecticidal crystal proteins which can tolerate temperatures higher than these values.29 0.120 X1 (g/min) X2 (m3/min) X3 (°C) X4 (MPa) 0. the Equation for SIW will be: 2 2 VSðSIWÞ ¼ 1010 ðÀ2:7 þ 3:5X1 À 5:6X1 þ 2:0X2 À 1:9X2 2 2 À 6: 9X4 Þ þ 1:7 Â 108 X3 À 4:8 Â 105 X3 ð 2Þ where VS. except for experiment no 18 in the case of SIW.0002) and. Likewise.10 MPa are the optimal values of independent variables for high spore count in the case of SIW. X2.75%) in the case of SIW was probably due to the high feed rate (0.Bioprocess Biosyst Eng (2011) 34:237–246 Table 2 Levels of the factors tested in the central composite design 241 Independent variables Symbol Levels of factors -2 -1 0 1 2 Starch industry wastewater Feed ﬂow rate Hot air aspiration ﬂow rate Inlet temperature Pulverization pressure Secondary hydrolyzed sludge Feed ﬂow rate Hot air aspiration ﬂow rate Inlet temperature Pulverization pressure X1 (g/min) X2 (m3/min) X3 (°C) X4 (MPa) 0.23 0.  who used the method of spray drying for Bt powders. the statistical analysis of the coefﬁcients of regression showed that: (i) linear coefﬁcients of the variables X1. Thus.40 0.26 0.0002). X3.57 190 0. the values of outlet temperatures were relatively lower than the temperature used for determination of the count of viable spores (80 °C). The independent effects of feed rate (X1).35 0.62 200 0. the linear coefﬁcients. The statistical analysis.94 for SIW and TH.35 0. for SIW and TH.29 g/min.51 m3/min. The variability in the response with the supposition of 92% for SIW and 94% for TH can be explained by this model. inlet temperature (X3) and pressure of air atomization (X4) on the number of spores are presented in Fig. similar to the ones obtained by Alamilla et al. X1. the analysis of the results on the basis of Eq. The linear effects and the interactions were not signiﬁcant in the case of two media. In the case of TH.52 180 0. R2 of the model represents the percentage of variation in the esponse which can be explained. moisture values were lower than 10%.16 Outlet temperature and moisture content It was observed that. inlet temperature. pressure of air atomization.110 0. The method of surface response was used to determine the optimum conditions for drying that gave high viable spore counts. b.075 g of powder used for the measurement of Tx and number of viable spores. This can be explained by the fact that high moisture content decreases the quantity of dry matter contained in 0. X2 = 0.7).7 9 108 CFU/mg in the initial fermented broth with a relatively high loss of 18%.49 170 0. aspiration rate. However. In the case of SIW. the regression model showed the same conﬁguration as noted for the quadratic effects (negative and signiﬁcant with p \ 0.44 165 0. X3 and X4 have statistically signiﬁcant negative effects (P = 0. (ii) quadratic coefﬁcients of 2 2 2 X2 1.060 0.54 175 0. The lack of ﬁt test is a measure of the failure of the model to represent data in the experimental region in which points were not included in the regression . (iii) coefﬁcient of interactions were not statistically signiﬁcant (P [ 0.070 0. X2.45 0. X4. R2 of 0. X2.125 0. produced less 123 .92 and 0.
6E?07 2.5E?07 4.29 0.075 0.1E?07 3.32 0.47 0.160 0. pressure of air atomization.47 0.32 0.242 Table 3 Results of experimental plan by central composite design for starch industry wastewater n° Independent variables X1 (g/min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 0.99 13.110 0. This condition gave high viable spore count of 1.1E?07 2.29 0.125 0. diet and conditions ð 3Þ where VS. aspiration rate.29 8.52 0.26 0. X2 ﬂow rate of hot air. respectively.62 0.2E?08 1. X2.075 0.32 0.110 0. number viable spore count in TH medium.87 3. X1.125 0.26 0.60 5. Entomotoxicity The analysis of the results of entomotoxicity for the two media was done by ANOVA with the regression models given by Eq.57 0.075 0.01 5.57 4.1E?07 2.29 0. following optimal values were obtained: X1 = 0.110 0.52 4.110 0.32 0.57 0.7E?07 2.12 4.52 0. such as the larval environment.13 4.075 0.49 m3/min.62 3. inlet temperature. X3.46 3.29 5.110 0.47 0.26 0.075 0. X4.1E?07 2.35 0.26 0.47 0.45 3.92 3. b) as compared with 123 .29 0.110 0.52 0.36 5.26 0.002 = P = 0. the Equation for TH will be: 2 2 VSðTHÞ ¼ 1010 ðÀ4:0 þ 1:2X1 À 1:2X1 þ 1:1X2 À 1:2X2 8 2 2 þ 3:2X4 À 18X4 Þ þ 3:9 Â 10 X8 À 1:2106 X3 1. With this model.3E?07 4.6E?07 1.47 0.52 3 Bioprocess Biosyst Eng (2011) 34:237–246 Dependent variables X3 (°C) 170 170 190 190 170 170 190 190 170 170 190 190 170 170 190 190 180 180 180 180 160 200 180 180 180 180 180 180 180 180 180 X4 (MPa) 0.47 0.84 3.6E?07 3.47 0. X3 inlet air temperature and X4 pressure of atomization of air signiﬁcant effects than in the case of SIW (0.57 0.4E?07 3.84 VS (CFU/mg) 1.52 0.1E?07 3.8E?08 2.29 0.125 0.14 4.110 0.1E?07 1. 3a.29 0.3E?07 1.26 0.3 9 108 CFU/mg (Fig.52 0.110 0.29 0.125 0.0E?07 2.25 5.2E?07 3.29 X2 (m /min) 0.12 5.9E?07 3. feed rate.125 0.52 0.27 3.125 0.29 0.57 0.7E?07 3.67 4. Hence.52 0.47 0.0E?07 3. These low coefﬁcients can be explained by the fact that the measurement of entomotoxicity involved other parameters.4E?08 Tx (9106 SBU/g) 554 610 499 480 443 671 234 222 610 720 554 530 554 523 582 388 831 111 659 610 554 388 610 610 665 665 720 655 637 776 677 X1 feed rate of pump.4E?07 2.26 0.68 3.52 0.32 0.45 g/min.26 0.1E?07 3. X3 = 170 °C and X4 = 0.29 0.02).52 0.110 Toutlet (°C) 44 44 52 52 65 70 73 75 42 43 45 46 51 53 71 73 58 45 46 62 40 58 56 55 54 53 53 53 53 54 55 M (%) 4.5 9 108 CFU/mg in the initial fermented broth of TH.7E?07 2.57 0.57 0.42 4.0E?07 2.096 MPa.57 0.53 3.32 0.59 9.29 0.6E?08 2.29 0.30 4.52 0.65 5.075 0. which gave a relatively high loss of 13%.75 4.3E?08 1.4E?08 2.32 0.110 0.125 0.110 0.9E?08 2.110 0.32 0.060 0.125 0.29 0.075 0.23 0.110 0. X2 = 0.42 0.2E?07 3. (1).25 5.29 8.075 0.57 0. This method showed that the regression models demonstrated lower coefﬁcient of determination (R2) of 57 and 58% for SIW and TH.52 0.52 0.
095 Toutlet (oC) 45 46 49 53 67 73 74 77 45 44 46 49 52 54 72 70 61 46 47 63 42 61 58 56 55 55 56 54 55 57 57 M (%) 3.99 4.50 0.45 3.44 0.45 0.40 0.54 0.71 3.50 0.79 VS (CFU/mg) 1.51 4.98 9 108 and 7.095 0.095 0.35 0.45 0. Hence.44 0.110 0.40 0.110 0.20 9 108 SBU/g for SIW and 5. X3 inlet air temperature and X4 pressure of atomization of air (temperature and external moisture) which can inﬂuence the actual obtained values.49 0.1E?07 2.55 0.0E?07 2.Bioprocess Biosyst Eng (2011) 34:237–246 Table 4 Results of experimental plan by central composite design for secondary hydrolyzed sludge n° Independent variables X1 (g/min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 0.49 0.49 0.45 0.44 0. the values of entomotoxicity were 7.095 0. This loss can be explained due to partial inactivation of various active components of Bt (spores.59 0.45 0.31 9 108 SBU/g in their respective fermented broths.4E?08 Tx (9106 SBU/g) 499 466 432 399 424 451 366 563 499 447 432 451 488 432 466 399 599 565 528 499 466 521 480 532 532 665 543 576 565 599 599 X1 feed rate of the pump.45 0.44 0.3E?08 9.49 3 243 Dependent variables X3 ( C) 165 165 175 175 165 165 175 175 165 165 175 175 165 165 175 175 170 170 170 170 160 180 170 170 170 170 170 170 170 170 170 o X4 (MPa) 0.9E?07 1.50 0.0E?07 1.5E?07 1.19 6.110 0.67 3. with the given optimal values by statistical analyses of the results of the spores.6E?06 1. The loss of entomotoxicity is higher in the case of SIW and it could be attributed to lower concentration of total solids in the fermented broth.93 4.59 4. enzymes) which are affected by the inlet temperature and pressure during spray drying.50 0. .4E?07 1.5E?07 6. insecticidal crystal proteins.8E?07 1.7E?07 1.095 0.54 0.6E?07 1.39 0.74 3.99 4.120 0.15 3. it was not necessary to determine the optimal conditions using these models.4E?07 9. Higher suspended solids concentration in hydrolyzed sludge may act as protective sheath against inactivation of active ingredients leading to lower Tx losses.085 0.40 0. In the light of the results of this work.54 0.45 0.9E?07 1.9E?07 1.54 0.49 0.73 5.13 4.37 4.50 0.095 0.44 0.4E?07 1. there was an entomotoxicity loss of 28% in the case of SIW and 18% in the case of TH.54 0.45 0.40 0.25 5. X2 ﬂow rate of hot air.49 0.085 0.45 0.44 0.49 0.44 0.7E?08 1.40 0. However.49 3.3E?07 1.45 4.7E?07 1.7E?07 2.085 0.110 0.49 0.095 0.44 0.45 0.45 0.56 3.67 9.49 0.12 4.95 7. the increase in the 123 .40 0.095 0.9E?07 1.095 0.49 0.6E?06 1.49 0.9E?07 1.75 6.085 0.095 0. On the other hand.40 0. lower solids concentration in SIW fermented broth resulted in poor protective sheath and longer evaporation time (due to higher water content) thus keeping the ingredients at high temperature causing higher loss of the active ingredients.40 0.97 5.25 3.085 0.35 4.11 3.45 X2 (m /min) 0.085 0.110 0.50 0. In fact.110 0.99 9 108 SBU/g for compared with 9.4E?08 1.65 3.50 0.75 4.49 0.085 0.50 0.89 4.6E?07 2.45 0.085 0.54 4.49 0. As the coefﬁcients were poor.1E?08 1. according to Vu et al.54 0.095 0.54 0.095 0.110 0.45 0.54 0.070 0. it was observed that there was not a large difference between the optimal conditions of two media (SIW 15 g/l and TH 25 g/l).6E?07 1.4E?08 1.095 0.110 0.9E?07 1.8E?07 1.
2 a Response surface of viable spores obtained by varying feed ﬂow (X1) rate and hot air ﬂow rate (X2) and keeping inlet temperature (X3) and air atomization pressure (X4) constant (case of SIW). Estimation of fermented broth volume for powder formulation and ﬁeld application By using these optimized results of spray drying. Table 5 demonstrates the volume of fermented broth of SIW and TH required for producing powder formulation which can be applied to treat 1 ha of balsam ﬁr. the volume of fermented broth required to produce powder product in order to treat 1 ha of balsam ﬁr was calculated and compared with the volume required for liquid product. viable spores and entomotoxicity can be increased in the powders of SIW by increasing the solids concentration of the fermentation medium. Therefore. respectively after spray drying due to the destruction of bioactive compounds during spray drying. The recom(1996–2001) in southwestern Que mended dosage of 30 BIU could be applied in lower application volumes (1. (2) 11% of the powder will be lost during industrial spray drying process 123 . In our earlier research. healthy stands against the spruce budworm was determined over a period of 6 years ´ bec .). Abies balsamea (L. b Response surface of viable spores obtained by varying the inlet temperature (X3) and air atomization pressure (X4) and keeping feed ﬂow (X1) and drying hot air ﬂow rate (X2) constant (case of TH) concentration of SIW up to 30 g/L enhanced the viable spores count and the entomotoxicity of the fermented broth. Following assumptions were considered in the calculation: (1) loss of Tx in dried powders of SIW and TH was 28 and 18%. Field efﬁcacy of Btk based biopesticides to specify application prescriptions for optimal protection of balsam ﬁr.5 l/ha) by using a high-potency product (20 BIU/l) or higher application volumes (2. Commercial Btk biopesticides products are normally based on international unit (IU) determined using cabbage looper larvae as target insect for bioassay . Thus. b Response surface of viable spores obtained by varying the inlet temperature (X3) and air atomization pressure (X4) and keeping feed ﬂow (X1) and hot air ﬂow rate (X2) constant (case of SIW) Fig. the SBU measured in this research was converted to IU by using the above factor of conversion.7 BIU/l) without affecting the ﬁeld efﬁcacy . 3 a Response surface of viable spores obtained by varying feed ﬂow (X1) rate and hot air ﬂow rate (X2) and keeping inlet temperature (X3) and air atomization pressure (X4) constant (case of TH).244 Bioprocess Biosyst Eng (2011) 34:237–246 Fig. we found that spruce budworm unit (SBU) determined against spruce budworm is 20–25% higher than IU .37 l/ha) of a lower potency product (12.
Powder formulation can offers advantages of being used in lesser amounts as compared to liquid formulations.2 6.5 g of solids were lost after centrifugation of fermented broth in liquid formulation. our previous research demonstrated 21–22 l of fermented broth of SIW (15 g/l total solids) was required to produce the ﬁnal liquid formulation (2.1 9. In powder formulation. personal communication).) for formulation .62 TH 25 0. 3. about 133. after centrifugation and mixing the pellet with supernatant.Bioprocess Biosyst Eng (2011) 34:237–246 Table 5 Volume of fermented broth required to produce powder formulation which can be applied to 1 ha of balsam ﬁr (30 BIU/ha) Fermentation medium Total solids concentration of 1 l of fermented broth (g) Tx of fermented broth (109 SBU/g) Tx of dried powder (109 SBU/g) Loss of Tx during spray drying (%)a Powder loss in the chamber wall of spray-drier (%) Total dried powder obtained from 1 l of fermented broth (g)b Total Tx obtained from 1 l of fermented broth after spray drying (109 SBU)c Total Tx obtained from 1 l of fermented broth after spray drying (109 BIU)d Total Tx of formulated powder from 1 l of fermented broth (BIU)e Volume of fermented broth required to produce formulated powder to treat 1 haf a b c 245 SIW 15 1 0. (3) storage at room temperature.73 0.5 g of total solids with total Tx of 96. the number of viable spores decreased compared with that of fermented broth due to the negative effects of operational parameters of spray drying.25 13. In liquid formulation. In the case of liquid formulation. such as residual delta-endotoxin. The views and opinions expressed in this article are those of authors. stickers. 4. Under the optimal conditions. after spray drying. f Conclusions in drying chamber wall and in transfer pipes of spray-drier (R. Acknowledgments The authors are sincerely thankful to the Natural Sciences and Engineering Research Council of Canada (Grants A4984. (2) requirement of less space for storage.5 4. phagostimulants. (4) spray drying can convert the entire fermented broth into solid form and does not require centrifugation step. For example.6 7. 123 . it is known that centrifugation of fermented broth could not recover all bioactive compounds. (5) standard application of Bt based biopesticides is 30 BIU/ha of balsam ﬁr . 1 l of suspended pellet with total solids concentration of 50 g/l and total Tx of 20. Low concentration of total solids of fermented broth caused a high loss of viable spores and entomotoxicity.2 l of suspension) to treat 1 ha of balsam ﬁr . 2.1–2.35 9. (3) Tx value of dried powder will be reduced by 25% due to the conversion from SBU into IU . In fact. about 83. 27] which was usually discarded. vegetative insecticidal proteins (Vips) [26. Lewandoskii. STP235071 and Canada Research Chair). The lost solids probably contained bioactive compounds incurring higher volumes of fermented broth for liquid formulation. and (5) supernatant obtained after centrifugation of fermented broth (in the case of liquid formulation) requires further treatment (wastewater treatment).72 28 11 13. the obtained products will have different total solids concentration. Kokou Adjalle is grateful ´ te ´ de Protection des Fore ˆ ts contre les Insectes et to SOPFIM (Socie Maladies) for the Smirnoff scholarship. (4) less chance of contamination due to less humidity. It is clear that powder formulation has some advantages as compared with liquid formulation such as: (1) requirement of less volume of fermented broth to produce formulated product to treat per ha of balsam ﬁr. In powder formulation. enzymes. IU values of formulated product were reduced to 22% due to the fortiﬁcation of additives (antimicrobial agents.1 3.9 9 109 SBU was obtained . spores. The volume of fermented broth required for powder formulation is less than that of liquid formulation (Table 5).3 Due to destruction of bioactive compounds during spray drying Calculated based on the loss of powder during spray drying Multiplying dried powder Tx with total dried powder obtained from 1 l of fermented broth Conversion of SBU to BIU  Tx will be reduced to 10% due to formulation of dried powder with formulated agents Based on standard application of Bt biopesticides (30 BIU/ha)  d e [26. 27].6 18 11 22. Thus.1 9 109 SBU was obtained. INRS-ETE and FQRNT (ENC) for ﬁnancial support. These compounds are soluble and present in the supernatant of centrifuged fermented broth Optimization of spray drying processes employing surface response methodology for the production of Bt powders from fermented broths of SIW and TH led to following conclusions: 1.4 10. Optimal values of the independent variables allowed high number of viable spores with higher determination coefﬁcients R2 [ 90% for the two media. with 10 l of fermented broth (15 g/l total solids) used for production of liquid or powder formulation. preservatives. Based on standard application of 30 BIU/ha. (4) Tx obtained after powder formulation will be reduced by about 10% due to the addition of additives required for formulation (dilution) . these bioactive compounds were recovered by spray drying.
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