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Abstract: Bacillus thuringiensis can be produced by a fermentative process using agro-industrial residues as substrates. The culture medium in solid state fermentation (SSF) is composed by moist nutrients. The water is a limiting factor in the SSF, differently from that in submerged fermentation. The solid culture medium must have a minimum moisture content to allow microbial growth but the concept of water availability in the substrate is best explained in terms of water activity (Aw). This study investigated the water activity influence on the solid state fermentation of Bacillus thuringiensis using rice husk, bran and grits as substrates. Three culture media were tested: (i) rice grits; (ii) rice grits and rice husk; (iii) rice bran and rice husk. In order to study hygroscopic characteristics of each medium, isotherms were constructed. For each culture medium, two processes were carried out: one with low Aw and the other with high Aw. The fermentations with B. thuringiensis were made in plastic bags containing the culture medium. The results showed differences in productivity. The best results were obtained for the medium composed by rice grits. The productivity((spores/g medium)/h) for the process using initial Aw of 0.620 was 3.0 ? 104 and for the initial Aw of 0.965 was 1.0 ? 106.
The use of chemical pesticides in agriculture has caused extensive damage to the environment since their high toxicity is harmful to beneficial insects, domestic animals, and to man. In addition, some species have already shown resistance problems. An alternative to pest control may be the use of pesticides or the rational combination of chemical and biological pesticides. There is a wide variety of microorganisms(fungi, bacteria, and viruses) that can act as entomopathogens in the biological control of pests. Among them we find Bacillus thuringiensis, which is a bacterium that has great potential, is effective against a wide range of insects, and is harmless to humans, animals, beneficial insects, and natural predators [1-5]
B. thuringiensis can be produced by fermentation processes using agro-industrial residues of low commercial value as substrates. Throughout the various stages in rice processing, from harvest to the production of graded, polished white rice, the following by-products and residues are produced: rice straw, husk, bran, and grit, which can be considered residues of great potential use as substrates [6-8].
Ecologically, the bio-utilization of these residues for pesticide production can be considered very important because besides being a solution to industries, these products do not harm the environment and will be used in agriculture, which provides raw materials for their production [9].
Among the techniques used in fermentation processes, the following can be highlighted: fermentation in liquid medium or submerged fermentation (SmF) and solid-state fermentation (SSF).
approximately 0.7 U.S. dollars. On the other hand, Sella et al. [13] published a manuscript on Bacillus atrophaeus spores’ production by solid-state fermentation. The authors used soybean molasses as substrate and sugarcane bagasse as support.
According to the literature, there are few studies on the influence of water activity on solid state fermentation, especially with B. thuringiensis. However, in order for microbial growth to occur, in addition to sources of carbon, nitrogen, and some salts, particularly for the SSF, a minimum amount of water required for the development of physiological activity of a microorganism should be taken into account, which varies according to type and variety. Bacteria, for example, require greater amounts of water than fungi and yeasts. Therefore, water in the SSF becomes a limiting factor in this process, which does not occur in the liquid fermentation, in which there is an abundant aqueous phase. In the solid culture medium, minimum moisture content is necessary for microbial growth; however, the concept of water available in the substrate for the use of microorganism is very different from moisture content, and it is better expressed in terms of water activity (Aw). The relationship between substrate moisture and its corresponding water activity at a given temperature is represented by curves called isotherms showing the hygroscopic characteristics of each material [11, 15-20].
The objective of this study is to investigate the influence of water activity (Aw) and hygroscopic characteristics of culture media on production of Bacillus thuringiensis by solid state fermentation.
Since the husk is the poorest substrate in nutrients, it can be used as an adjuvant in the process, for example, along with fine grained substrates that have a tendency to remain aggregated and thereby assist in the improvement of mass transfer between its particles. This was done in this study when bran and ground rice grits were used. Rice husk could also be used as a support for liquid substrates rich in nutrients.
With regard to size, rice grits are considered medium sized particles, rice bran, husk, and ground grits are considered fine grained particles. Rice grit grains have an average diameter of 1.61 mm, whereas the average diameter of ground grit particles is 0.68 mm; for husk it is 0.70 mm, and for bran it is 0.79 mm.
The water adsorption curves obtained for the three compositions of culture medium studied are illustrated in Fig. 1. Those curves were obtained from the substrates without addition of water and that do not undergo any drying process. The only treatment those substrates underwent, in all cases, was sterilization by autoclaving at 121 °C for 20 minutes. It can be noted that the substrates have different water activity and moisture content showing that the materials to be used in SSF should be studied taking into account their hygroscopic properties. The differences observed between the curves shows a change in the characteristics
which indicates a smaller amount of water in the outermost layer, but with increasing Aw values, it falls between those of the other two substrates.
When constructing these curves, it was found that to obtain similar Aw values, the amount of water added to each composition of culture medium was not the same. For the rice bran and husk mixture, the required amount of water is twice as much as that for the rice grits to obtain an Aw value of approximately 0.960. This shows once again that if the chemical composition of each substrate is different, their hygroscopic characteristics will also be different.
The Aw value also indicates the potential of water that is directly connected with the energetic state of water in organic substrates. The water potential of a solid substrate is essentially defined as the sum of two components: osmotic potential (related to the concentration and physicochemical properties of solutes) and matric potential (related to adsorption and capillarity of the solid matrix). For the equilibrium between the liquid, solid, and gaseous phases, water potential may be related to relative humidity (RH) of the gas phase or the Aw of the substrate [11, 16-18, 27, 28].
According to Murthy et al. [11], water activity (Aw) sets the amount of unbound water available in the immediate surroundings of the microorganism; therefore it better defines the limits of growth of microorganisms because they do not recognize the moisture content of the material, but rather the amount of water available, whose variation depends on the solute. In solid state culture, microbial cells grow either on the surface or within the particle of solid matrix, where water is sufficient to sustain growth [29].
Until recently, Aw was considered as a physicochemical parameter in two scientific disciplines: physical chemistry and food microbiology. According to the former, it was considered the thermodynamic concentration of water; and according to the latter, it was considered the growth limit of food spoiling microorganisms. The reason why water activity is used instead of moisture content in
microbiology is that it has been shown that microorganisms which deteriorate food can grow in a wide range of moisture levels [30]. Hahn-H?gerdal[18] stated that Aw should be seen as another parameter of the physicochemical process (such as pH, PO2, and temperature), which under certain conditions act synergistically with other parameters, and in some cases can determine the progress of some biological, microbiological, or enzymatic processes.
In the SSF, water should be available for microbial growth and biochemical activities to occur. Every microorganism has a minimum Aw to carry out its metabolic activities. For example, the optimum Aw for fungi is around 0.7, for yeast it is 0.8, and for bacteria it is 0.9 [31]. A small variation in these optimum values causes major disturbance in the growth and metabolism of microorganisms [32].
Corona et al. [33] carried out several studies on Aw and microbial growth. For example, in Penicillium roqueforti, Aw is the critical variable for growth and spore production in SSF. Some studies report that the optimum Aw for growth of Trichoderma viride ranges between 0.99 and 0.992, while spore production is maximized at Aw 0.98. On the other hand, the optimum Aw for cyclopeptide production in Metarhizium anisopliae is 0.921 although variations according to media composition were observed. Results found for Gibberella fujikuroi growth rates at different Aw levels minimal values indicated that the minimal Aw value that supported growth was approximately 0.9. Growth rate increased continuously up to an Aw value of 0.995, and then it decreased slowly. The optimum growth for Gibberella fujikuroi was obtained between Aw values of 0.985 and 0.995.
The induction of sporulation of some genera of fungi and bacteria occurs in response to several factors including: availability of nutrients, unfavorable pH, and osmotic stress, for example, changes in the Aw environment. Bacterial endospores and some types of fungal spores are characterized by the need for special conditions in order to start germination and development. Among those needs, according to Beuchat [31], are optimal values of Aw. Grajek and Gervais [17] found that Aw strongly affects the sporogenesis of Trichoderma viride. Their results proved that the Aw can be a limiting parameter in the culture development.
Lower water activity result in lower mass transfer rates and little water available for microorganisms, and it may be responsible for incomplete conversion of substrate to biomass [11]. However, the behavior of isotherms provides important information regarding the greater or lesser water availability within the range studied (0.6 to 0.9). It is evident that the free water, represented by the second region of the isotherms, will be more readily available, whereas for smaller Aw values, represented by the first region of the isotherms, the water will become available as the layers are used and change the adsorption thermodynamic balance, i.e., the water release rate will be lower. This can be beneficial for microorganism growth.
Fig. 2 shows the growth curves of B. thuringiensis based on spore production.
Based on the isotherms, it can be said that in experiments 3 and 4 (performed with ground rice grits and rice husk) the speed of the initial production of
which losses can occur and thus, at the end of the process it can have the same or lower concentration of spores than that of the product obtained in this study. It is worth mentioning that costs with energy in the drying process may make the process economically unfeasible. Therefore, the ideal condition is to work with the necessary moisture only to avoid the need to remove excess water, minimize the costs with energy in the recovery of the product, and produce less effluents; concerning the present study, the production time was reduced considerably (from 112 h to 36 h).
Hardman (Ed), Water and Food Quality, Elsevier Applied Science, London, 1989, p. 1.
[17] W. Grajek, P. Gervais, Effect of the sugar-beet pulp water activity on the solid-state culture of Trichoderma viride TS, Applied Microbiology and Biotechnology 26 (1987) 537-541.
[18] B. Hahn-H?gerdal, Water activity: A possible external regulator in biotechnical processes, Enzyme Microbiology and Technology 8 (1986) 322-327.
[19] B.K. Lonsane, N.P. Ghildyal, S. Budiatman, J. Ramakrishna, Engineering aspects of solid state fermentation, Enzyme Microbiology and Technology 7(1985) 258-265.
[20] W.J. Scott, Water relations of food spoilage microorganisms, Advances on Food Research 7 (1957) 83-127.
[21] W. Horwitz, A. Senzel, H. Reynolds, D.L. Park, Official Methods of Analysis of the Association of Official Analytical Chemists, Association of Official Analytical Chemists, Washington, 1990.
[22] E.C. Bligh, W.J. Dyer, A rapid method of total lipid: Extraction and purification, Canadian Journal of Biochemistry Physiology 37 (1959) 911-917.
[23] O. Zenebon, N.S. Pascuet, P. Tiglea, Métodos Físico-químicos para Análise de Alimentos, Instituto Adolfo Lutz, S?o Paulo, 2008.
[24] A.H. Landrock, B.E. Proctor, A new graphical interpolation for obtaining humidity equilibria data, with special reference to its role in food packaging studies, Food Technology 5 (1951) 332-337.
[25] P.J. Thompson, K.E. Stevenson, Mesophilic sporeforming aerobes, in: M. Speck (Ed.), Compendium of Methods of Microbiological Examinations of Foods, American Public Health Association, Washington, 1984, p. 211.
[26] G.C. Dors, R.H. Pinto, E. Badiale-Furlong, Influência das condi??es de parboiliza??o na composi??o química do arroz, Ciência e Tecnologia de Alimentos 29 (2009) 219-224.
[27] P. Gervais, P. Molin, The role of water in solid-state fermentation, Biochemical Engineering Journal 13 (2003) 85-101.
[28] P. Gervais, M. Bensoussan, W. Grajek, Water activity and water content: Comparative effects on the growth of P. roqueforti on solid substrate, Applied Microbiology and Biotechnology 27 (1988) 389-392.
[29] C. Krishna, Solid-state fermentation systems—an overview, Critical Reviews on Biotechnology 25(2005) 1-30.
[30] D.A.A. Mossel, Water relations of foods, in: R.B. Duckworth (Ed.), Food Science and Technology, Academic Press, London, 1975, p. 347.
Biochemistry 40 (2005) 2655-2658.
[34] I.O. Moraes, L.H. Pelizer, D.M.F. Capalbo, Estudo da atividade de água e fermenta??o semi-sólida do Bacillus thuringiensis em arroz, Simpósio Latino Americano de Ciência de Alimentos, Campinas (1995) 37.
[35] M. Loncin, Basic principles of moisture equilibria, in: S. Goldblith, L. Rey (Eds.), Freeze-drying and Advanced Food Technology, Academic Press, New York, 1975, p. 599.
[36] K.D. Ross, Estimation of water activity in intermediate moisture foods, Food Technology 29 (1975) 26-34.
The use of chemical pesticides in agriculture has caused extensive damage to the environment since their high toxicity is harmful to beneficial insects, domestic animals, and to man. In addition, some species have already shown resistance problems. An alternative to pest control may be the use of pesticides or the rational combination of chemical and biological pesticides. There is a wide variety of microorganisms(fungi, bacteria, and viruses) that can act as entomopathogens in the biological control of pests. Among them we find Bacillus thuringiensis, which is a bacterium that has great potential, is effective against a wide range of insects, and is harmless to humans, animals, beneficial insects, and natural predators [1-5]
B. thuringiensis can be produced by fermentation processes using agro-industrial residues of low commercial value as substrates. Throughout the various stages in rice processing, from harvest to the production of graded, polished white rice, the following by-products and residues are produced: rice straw, husk, bran, and grit, which can be considered residues of great potential use as substrates [6-8].
Ecologically, the bio-utilization of these residues for pesticide production can be considered very important because besides being a solution to industries, these products do not harm the environment and will be used in agriculture, which provides raw materials for their production [9].
Among the techniques used in fermentation processes, the following can be highlighted: fermentation in liquid medium or submerged fermentation (SmF) and solid-state fermentation (SSF).
approximately 0.7 U.S. dollars. On the other hand, Sella et al. [13] published a manuscript on Bacillus atrophaeus spores’ production by solid-state fermentation. The authors used soybean molasses as substrate and sugarcane bagasse as support.
According to the literature, there are few studies on the influence of water activity on solid state fermentation, especially with B. thuringiensis. However, in order for microbial growth to occur, in addition to sources of carbon, nitrogen, and some salts, particularly for the SSF, a minimum amount of water required for the development of physiological activity of a microorganism should be taken into account, which varies according to type and variety. Bacteria, for example, require greater amounts of water than fungi and yeasts. Therefore, water in the SSF becomes a limiting factor in this process, which does not occur in the liquid fermentation, in which there is an abundant aqueous phase. In the solid culture medium, minimum moisture content is necessary for microbial growth; however, the concept of water available in the substrate for the use of microorganism is very different from moisture content, and it is better expressed in terms of water activity (Aw). The relationship between substrate moisture and its corresponding water activity at a given temperature is represented by curves called isotherms showing the hygroscopic characteristics of each material [11, 15-20].
The objective of this study is to investigate the influence of water activity (Aw) and hygroscopic characteristics of culture media on production of Bacillus thuringiensis by solid state fermentation.
Since the husk is the poorest substrate in nutrients, it can be used as an adjuvant in the process, for example, along with fine grained substrates that have a tendency to remain aggregated and thereby assist in the improvement of mass transfer between its particles. This was done in this study when bran and ground rice grits were used. Rice husk could also be used as a support for liquid substrates rich in nutrients.
With regard to size, rice grits are considered medium sized particles, rice bran, husk, and ground grits are considered fine grained particles. Rice grit grains have an average diameter of 1.61 mm, whereas the average diameter of ground grit particles is 0.68 mm; for husk it is 0.70 mm, and for bran it is 0.79 mm.
The water adsorption curves obtained for the three compositions of culture medium studied are illustrated in Fig. 1. Those curves were obtained from the substrates without addition of water and that do not undergo any drying process. The only treatment those substrates underwent, in all cases, was sterilization by autoclaving at 121 °C for 20 minutes. It can be noted that the substrates have different water activity and moisture content showing that the materials to be used in SSF should be studied taking into account their hygroscopic properties. The differences observed between the curves shows a change in the characteristics
which indicates a smaller amount of water in the outermost layer, but with increasing Aw values, it falls between those of the other two substrates.
When constructing these curves, it was found that to obtain similar Aw values, the amount of water added to each composition of culture medium was not the same. For the rice bran and husk mixture, the required amount of water is twice as much as that for the rice grits to obtain an Aw value of approximately 0.960. This shows once again that if the chemical composition of each substrate is different, their hygroscopic characteristics will also be different.
The Aw value also indicates the potential of water that is directly connected with the energetic state of water in organic substrates. The water potential of a solid substrate is essentially defined as the sum of two components: osmotic potential (related to the concentration and physicochemical properties of solutes) and matric potential (related to adsorption and capillarity of the solid matrix). For the equilibrium between the liquid, solid, and gaseous phases, water potential may be related to relative humidity (RH) of the gas phase or the Aw of the substrate [11, 16-18, 27, 28].
According to Murthy et al. [11], water activity (Aw) sets the amount of unbound water available in the immediate surroundings of the microorganism; therefore it better defines the limits of growth of microorganisms because they do not recognize the moisture content of the material, but rather the amount of water available, whose variation depends on the solute. In solid state culture, microbial cells grow either on the surface or within the particle of solid matrix, where water is sufficient to sustain growth [29].
Until recently, Aw was considered as a physicochemical parameter in two scientific disciplines: physical chemistry and food microbiology. According to the former, it was considered the thermodynamic concentration of water; and according to the latter, it was considered the growth limit of food spoiling microorganisms. The reason why water activity is used instead of moisture content in
microbiology is that it has been shown that microorganisms which deteriorate food can grow in a wide range of moisture levels [30]. Hahn-H?gerdal[18] stated that Aw should be seen as another parameter of the physicochemical process (such as pH, PO2, and temperature), which under certain conditions act synergistically with other parameters, and in some cases can determine the progress of some biological, microbiological, or enzymatic processes.
In the SSF, water should be available for microbial growth and biochemical activities to occur. Every microorganism has a minimum Aw to carry out its metabolic activities. For example, the optimum Aw for fungi is around 0.7, for yeast it is 0.8, and for bacteria it is 0.9 [31]. A small variation in these optimum values causes major disturbance in the growth and metabolism of microorganisms [32].
Corona et al. [33] carried out several studies on Aw and microbial growth. For example, in Penicillium roqueforti, Aw is the critical variable for growth and spore production in SSF. Some studies report that the optimum Aw for growth of Trichoderma viride ranges between 0.99 and 0.992, while spore production is maximized at Aw 0.98. On the other hand, the optimum Aw for cyclopeptide production in Metarhizium anisopliae is 0.921 although variations according to media composition were observed. Results found for Gibberella fujikuroi growth rates at different Aw levels minimal values indicated that the minimal Aw value that supported growth was approximately 0.9. Growth rate increased continuously up to an Aw value of 0.995, and then it decreased slowly. The optimum growth for Gibberella fujikuroi was obtained between Aw values of 0.985 and 0.995.
The induction of sporulation of some genera of fungi and bacteria occurs in response to several factors including: availability of nutrients, unfavorable pH, and osmotic stress, for example, changes in the Aw environment. Bacterial endospores and some types of fungal spores are characterized by the need for special conditions in order to start germination and development. Among those needs, according to Beuchat [31], are optimal values of Aw. Grajek and Gervais [17] found that Aw strongly affects the sporogenesis of Trichoderma viride. Their results proved that the Aw can be a limiting parameter in the culture development.
Lower water activity result in lower mass transfer rates and little water available for microorganisms, and it may be responsible for incomplete conversion of substrate to biomass [11]. However, the behavior of isotherms provides important information regarding the greater or lesser water availability within the range studied (0.6 to 0.9). It is evident that the free water, represented by the second region of the isotherms, will be more readily available, whereas for smaller Aw values, represented by the first region of the isotherms, the water will become available as the layers are used and change the adsorption thermodynamic balance, i.e., the water release rate will be lower. This can be beneficial for microorganism growth.
Fig. 2 shows the growth curves of B. thuringiensis based on spore production.
Based on the isotherms, it can be said that in experiments 3 and 4 (performed with ground rice grits and rice husk) the speed of the initial production of
which losses can occur and thus, at the end of the process it can have the same or lower concentration of spores than that of the product obtained in this study. It is worth mentioning that costs with energy in the drying process may make the process economically unfeasible. Therefore, the ideal condition is to work with the necessary moisture only to avoid the need to remove excess water, minimize the costs with energy in the recovery of the product, and produce less effluents; concerning the present study, the production time was reduced considerably (from 112 h to 36 h).
Hardman (Ed), Water and Food Quality, Elsevier Applied Science, London, 1989, p. 1.
[17] W. Grajek, P. Gervais, Effect of the sugar-beet pulp water activity on the solid-state culture of Trichoderma viride TS, Applied Microbiology and Biotechnology 26 (1987) 537-541.
[18] B. Hahn-H?gerdal, Water activity: A possible external regulator in biotechnical processes, Enzyme Microbiology and Technology 8 (1986) 322-327.
[19] B.K. Lonsane, N.P. Ghildyal, S. Budiatman, J. Ramakrishna, Engineering aspects of solid state fermentation, Enzyme Microbiology and Technology 7(1985) 258-265.
[20] W.J. Scott, Water relations of food spoilage microorganisms, Advances on Food Research 7 (1957) 83-127.
[21] W. Horwitz, A. Senzel, H. Reynolds, D.L. Park, Official Methods of Analysis of the Association of Official Analytical Chemists, Association of Official Analytical Chemists, Washington, 1990.
[22] E.C. Bligh, W.J. Dyer, A rapid method of total lipid: Extraction and purification, Canadian Journal of Biochemistry Physiology 37 (1959) 911-917.
[23] O. Zenebon, N.S. Pascuet, P. Tiglea, Métodos Físico-químicos para Análise de Alimentos, Instituto Adolfo Lutz, S?o Paulo, 2008.
[24] A.H. Landrock, B.E. Proctor, A new graphical interpolation for obtaining humidity equilibria data, with special reference to its role in food packaging studies, Food Technology 5 (1951) 332-337.
[25] P.J. Thompson, K.E. Stevenson, Mesophilic sporeforming aerobes, in: M. Speck (Ed.), Compendium of Methods of Microbiological Examinations of Foods, American Public Health Association, Washington, 1984, p. 211.
[26] G.C. Dors, R.H. Pinto, E. Badiale-Furlong, Influência das condi??es de parboiliza??o na composi??o química do arroz, Ciência e Tecnologia de Alimentos 29 (2009) 219-224.
[27] P. Gervais, P. Molin, The role of water in solid-state fermentation, Biochemical Engineering Journal 13 (2003) 85-101.
[28] P. Gervais, M. Bensoussan, W. Grajek, Water activity and water content: Comparative effects on the growth of P. roqueforti on solid substrate, Applied Microbiology and Biotechnology 27 (1988) 389-392.
[29] C. Krishna, Solid-state fermentation systems—an overview, Critical Reviews on Biotechnology 25(2005) 1-30.
[30] D.A.A. Mossel, Water relations of foods, in: R.B. Duckworth (Ed.), Food Science and Technology, Academic Press, London, 1975, p. 347.
Biochemistry 40 (2005) 2655-2658.
[34] I.O. Moraes, L.H. Pelizer, D.M.F. Capalbo, Estudo da atividade de água e fermenta??o semi-sólida do Bacillus thuringiensis em arroz, Simpósio Latino Americano de Ciência de Alimentos, Campinas (1995) 37.
[35] M. Loncin, Basic principles of moisture equilibria, in: S. Goldblith, L. Rey (Eds.), Freeze-drying and Advanced Food Technology, Academic Press, New York, 1975, p. 599.
[36] K.D. Ross, Estimation of water activity in intermediate moisture foods, Food Technology 29 (1975) 26-34.