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Brazilian Journal of Chemical Engineering

versão impressa ISSN 0104-6632versão On-line ISSN 1678-4383

Braz. J. Chem. Eng. v.19 n.2 São Paulo abr.jun. 2002 



C.L.Luna1, R.L.R.Mariano2 and A.M.Souto-Maior1,*
1Departamento de Antibióticos, Universidade Federal de Pernambuco,
Cidade Universitária, CEP 50.670-901, Recife - PE, Brazil.
2Departamento de Agronomia/Fitossanidade, Universidade Federal Rural de Pernambuco,
Dois Irmãos, CEP 52.071-900, Recife - PE, Brazil


(Received: February 10, 2002 ; Accepted: April 16, 2002 )



Abstract - Of nine epiphytic isolates of the genus Bacillus, only B. subtilis R14, B. pumilus C116, B. megaterium pv. cerealis RAB7, and B. cereus C210 showed antibiotic activity when tested in vitro against the bacterium Xanthomonas campestris pv. campestris LFR-3. Aiming product a biocontrol agent for black rot in crucifers, the production of B. subtilis R14 was evaluated in a batch bioreactor. Rapid growth was observed in a medium containing molasses and yeast extract as C-source and N-source, respectively. During the exponential growth phase, the specific growth rate was 1.2 h-1. A quick sporulation was also observed in a C/N well-balanced medium. After the sporulation phase, maximum viable spore concentrations around 109 CFU/mL were obtained. Preliminary sedimentation tests at different pH values showed better biomass separation efficiencies at low pH values.
Keywords: biocontrol agent, antibiotic activity, epiphytic, genus Bacillus, bacterium Xanthomonas.




Increasing the productivity of plants by improving their phytosanitary state with sustainability has been a great challenge for mankind in modern society. This objective has been achieved in many cases through the use of resistant varieties and chemical control. However, for some diseases, there is no source of resistance or new strains of pathogens, capable of infecting resistant varieties, may arise. Moreover, the indiscriminate use of agrochemicals leads to degradation of the ecosystem, may induce pathogen resistance to the pesticide, and may cause human and animal health problems (Huang, 1997).

Alternative strategies for disease management are necessary and include the use of bacteria that show benefic effects on plants, known as plant growth-promoting rhizobacteria (PGPR). The positive effects of PGPR are normally divided into two categories: growth promotion and biological control (Kloepper, 1997). Of the PGPR, species of the genus Bacillus have shown promising results for the growth promotion of several cultures and the biological control of various plant pathogens (Mariano et al., 1997). This occurs specially due to the capacity of Bacillus spp. to form spores, which survive and remain metabolically active under harsh environmental conditions (Rodgers, 1989), making them appropriate for the formulation of stable and viable products (Kloepper, 1997).

One of the most efficient biocontrol agents is B. subtilis, which shows biological activity against several phytopatogenic bacteria and fungi. This antagonism has been attributed to the production of peptide antibiotics (McKeen et al., 1986; Loeffler et al., 1986) and to the capacity for colonizing the plant. Recently, the use of Bacillus spp. has been studied for control of the most important bacterial disease in the crucifers (e.g., cabbage, kale, and radish), known as black rot, which is caused by the bacterium Xanthomonas campestris pv. campestris. In greenhouse experiments, Assis et al. (1996) observed that B. subtilis R14, isolated from the surface of kale leaves, completely controlled the disease on cabbage when tested against three strains of X. campestris pv. campestris in all application periods tested, suggesting its utilization as a preventive treatment for the disease. Besides, a 73% reduction in the disease in field tests corroborated the efficiency of this same isolate in controlling cabbage black rot (Assis et al., 1997).

The genus Bacillus includes a variety of important species used in the fermentation industry. Bacillus spp. are nonpathogenic, good secretors of proteins and metabolites, and easy to cultivate. Products currently available commercially include enzymes, antibiotics and insecticides. The topics addressed by this work were in vitro evaluation of the antimicrobial activity of nine epiphytic Bacillus isolates against X. campestris pv. campestris, a study of the production of B. subtilis R14 in a batch bioreactor using a molasses medium, and a preliminary study of separation of biomass from the fermentation broth by sedimentation.




The Bacillus spp. used were B. subtilis R14, B. megaterium pv. cerealis RAB7, B. megaterium pv. cerealis C211, B. pumilus C116, Bacillus sp RAB9, B. cereus C240, Bacillus sp C11, B. cereus C210, and Bacillus sp C21. These organisms were isolated from crucifers. Xanthomonas campestris pv. campestris LFR-3 was used as the test microorganism for determination of in vitro antibiotic activity. All microorganisms belong to the Laboratory of Phytobacteriology of the Department of Agronomy at the Federal Rural University of Pernambuco, Brazil.

Culture Media

During the experiments, Bacillus spp. were preserved by subculturing on a NA (nutrient agar) medium: meat extract, 3 g/L; meat peptone, 10 g/L; agar, 20 g/L. For X. campestris pv. campestris LFR-3, the medium used was YMA (yeast-malt agar): glucose, 10 g/L; peptone, 5 g/L; yeast extract, 3 g/L; malt extract, 3 g/L. Fermentations of B. subtilis R14 were carried out in molasses media, which are described in Table 1.



Experimental Procedures

For preliminary fermentation tests, the inoculum was grown in a test tube containing nutrient broth (NA without agar) at 30° C for 24 hours. This culture (2.5 mL) was transferred to a 250mL shaking flask, containing 25 mL of medium M1 or M2, which was incubated for 12 hours at 30° C and 200 rpm. Media with components sterilized together and separately were used.

Fermentations for spore production were carried out in a 5L bioreactor (BIOFLO III, New Brunswick Scientific) at 37° C and 700 rpm. Pre-inoculum was grown in a 250mL shaking flask, containing 25 mL of medium M3 or M4, for 12 hours at 30° C and 200 rpm. The culture was transferred to a Fernbach flask, containing 300 mL of medium M3 or M4, and after 24 hours at 30° C and 200 rpm, was inoculated in the bioreactor.

To study the influence of pH on biomass sedimentation, the pH values of broth fermentation samples were adjusted to 3.0, 5.0, and 7.0 with addition of 15 M HCl. Sedimentation tests were performed in a 100 mL graduated cylinder, containing 75 mL of the fermentation broth. Sedimentation efficiency was determined according to the following equation:

where XT and XS are the dry weight concentrations of the suspension (total biomass) and of the supernatant, respectively. To evaluate biomass concentration in the supernatant, samples were harvested when sediment reached 20% of the total volume (15 mL).

Analytical Methods

The agar diffusion method was used to measure the antibiotic activity of Bacillus spp. Blocks of NA containing Bacillus spp., grown at 30° C or 37° C for 48 hours, were placed over Petri dishes containing YMA that had been previously inoculated with X. campestris pv. campestris. The dishes were incubated at 28° C and after 24 hours the inhibition halos were measured.

Viable cell and spore concentrations were determined as CFU (colony-forming units) on the NA medium before and after thermal shock at 80° C for 12 minutes, respectively. Biomass concentration was determined as dry weight after filtering samples through millipore membranes and drying at 80° C for 24 hours. Reducing sugars in the filtrate were assayed using the DNSA (dinitrosalicylic acid) method (Miller, 1959). The broth filtrate activity was measured by the agar diffusion method, using a 6mm paper disc containing 20 m L of sample, under the same conditions as those described above for Bacillus spp. antibiotic activity tests.



Antimicrobial Activity of Bacillus spp.

The results of antibiotic activity tests, which are the averages of four repetitions, are presented in Figure 1. From the nine Bacillus tested, only B. pumilus C116, B. megaterium pv. cerealis RAB7, B. subtilis R14, and B. cereus C210 showed antibiotic activity.

While testing 32 epiphytic Bacillus against X. campestris pv. campestris on kale, Assis et al. (1996) showed that strains R14, C210, and C240 were able to achieve 100% control of the disease in two greenhouse experiments. In a third experiment, B. subtilis R14 also showed 100% control in three periods of application: three days before, simultaneously, and three days after inoculation with X. campestris pv. campestris. Besides, in field tests R14 as well as C210, C116, and C240 were also able to control this same bacterium on kale, in different periods of application and different kale cultivars (Assis et al., 1997). Correlating the results obtained in vitro in this work with those in vivo results reported in the literature, it can be found that at least isolates R14 and C210, which consistently control crucifers black rot disease in field tests, could have antibiosis as one mechanism of action. However, other mechanisms may also be involved (Kloepper, 1997).

Due to its performance in vivo and in vitro, B. subtilis R14 was selected for studies on spore production and separation.



Preliminary Fermentation Experiments

The aim of these fermentation experiments in shaking flasks was a preliminary study of the growth of B. subtilis R14 on a molasses-based medium. Media M1 and M2 - having, respectively, yeast extract and urea as nitrogen sources - were used. Whose experiments were carried out using media components had been sterilized together or separately to check for the occurrence of undesirable reactions when all components were autoclaved together (Cote and Gherna, 1994).

The results for these fermentations, which are averages of duplicates, are presented in Table 2. The data show that the different autoclaving conditions had no apparent negative effect on biomass accumulation. Table 2 also shows the suspended solid contents of the media, which are relatively low. This characteristic is important when one considers the biomass separation process and product formulation.

The formulation of media M1 and M2 was such that the amounts of molasses, nitrogen and phosphorous in their compositions were enough to produce a final biomass concentration around 5 g/L. The biomass obtained in 12 hours using yeast extract was more than three times that obtained using urea as the nitrogen source, indicating a much faster growth rate. This is not surprising since yeast extract is a rich source of amino acids, vitamins, and other growth factors.



Production of B. subtilis R14

Media with yeast extract as the nitrogen source were used for biomass production in the bioreactor. Figures 2 to 4 show the results for production of B. subtilis R14 in medium M3. In Figure 2, a rapid growth rate can be observed during the exponential phase, which ended after four hours of fermentation, when reducing sugars were still present in the bioreactor. A decrease in biomass after 10 hours can be associated with cell lyses and sporulation, when a residual amount of reducing compounds can still be observed, but which were not fermentable by the microorganism and remained in the medium throughout fermentation. After 24 hours of fermentation, a maximum amount of spores, around 109CFU, had already been attained, when fermentable reducing sugars had already been absent from the medium for about 14 hours (Figure 3). Figure 4 shows the variations in pH and dissolved oxygen (%DO) during fermentation. The rapid decrease in pH and %DO before 3 hours is associated with the exponential growth phase of the microorganism. Deceleration of growth is followed by increases in both pH and %DO. A second phase of decrease occurs while fermentable sugars are still being assimilated. A later rise in pH may be associated with sporulation.

Based on the results obtained above, the fermentation medium was reformulated. The ratio between molasses and yeast extract was reduced in medium M4, since sporulation is intensified after exhaustion of fermentable sugars. Figure 5 shows the results obtained for biomass accumulation and sugar consumption in medium M4. The results are similar during the exponential phase of growth as expected, and in this case the amount of samples taken allowed determination of the maximum specific growth rate, m max. In Figure 6, the plot of biomass concentration, X, versus time is shown for the exponential phase and the m max value presented. Sporulation increased after 6 hours when fermentable sugars were completely depleted (Figure 7). After 10 hours of fermentation, a spore concentration around 108 had already been attained. As predicted, production time could be considerably reduced in this case.

No antibiotic activity against X. campestris pv. campestris was detected in the fermentation broth sample filtrates during the fermentations in media M3 and M4. This could be expected since conditions favorable to growth are normally unfavorable to secondary metabolite production.








Biomass Separation by Sedimentation

The curves in Figure 8 show the influence of fermentation broth pH on biomass sedimentation.

The highest value obtained for sedimentation efficiency was 92% at pH 3.0, decreasing to 45% and 18% at pH 5.0 and pH 7.0, respectively (Table 3). When studying the sedimentation of B. sphaericus 2362, Luna (1999) also found the best results at a pH value of 3.0. Further studies are necessary to verify the mechanism involved in this separation process.





B. subtilis R14, B. megaterium pv. cerealis RAB7, B. pumilus C116, and B. cereus C210 show antibiotic activity in vitro against the bacterium X. campestris pv. campestris, the causative agent of black rot in crucifers. Spores of B. subtilis R14, which consistently control black rot disease in field tests, may be produced in a batch bioreactor using a molasses-based medium for the purpose of biocontrol. In a C/N well-balanced medium, production around 108 CFU/mL can be achieved in 10 hours of fermentation. Biomass separation from fermentation broth by sedimentation is enhanced when the pH value is decreased from neutral to acid.



The authors are grateful to Olho D’Água Sugar Industry, which kindly provided the molasses used in this work. They would also like to express their gratitude to Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for the financial support received.



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