Biotechnol Prog 2005, 21, 1026−1031 1026 ARTICLES A Procedure for High-Yield Spore Production by Bacillus subtilis Sandra M Monteiro,†,‡ Joa˜ o J Clemente,† Adriano O Henriques,‡ Rui J Gomes,† Manuel J Carrondo,†,‡,§ and Anto´ nio E Cunha*,†,‡ Instituto de Biologia Experimental e Tecnolo´gica (IBET), Apartado 1, P-2781-901 Oeiras, Portugal, Instituto de Tecnologia Quı´mica e Biolo´gica (ITQB), Universidade Nova de Lisboa, Apartado 127, P-2781-901 Oeiras, Portugal, and Laborato´rio de Engenharia Bioquı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Monte da Caparica, P-2829-516 Caparica, Portugal Bacillus subtilis spores have a number of potential applications, which include their use as probiotics and competitive exclusion agents to control zoonotic pathogens in animal production The effect of cultivation conditions on Bacillus subtilis growth and sporulation was investigated in batch bioreactions performed at a 2-L scale Studies of the cultivation conditions (pH, dissolved oxygen concentration, and media composition) led to an increase of the maximum concentration of vegetative cell from 2.6 × 109 to 2.2 × 1010 cells mL-1 and the spore concentration from 4.2 × 108 to 5.6 × 109 spores mL-1 A fed-batch bioprocess was developed with the addition of a nutrient feeding solution using an exponential feeding profile obtained from the mass balance equations Using the developed feeding profile, starting at the middle of the exponential growth phase and finishing in the late exponential phase, an increase of the maximum vegetative cell concentration and spore concentration up to 3.6 × 1010 cells mL-1 and 7.4 × 109 spores mL-1, respectively, was obtained Using the developed fed-batch bioreaction a 14-fold increase in the concentration of the vegetative cells was achieved Moreover, the efficiency of sporulation under fed-batch bioreaction was 21%, which permitted a 19-fold increase in the final spore concentration, to a final value of 7.4 × 109 spores mL-1 This represents a 3-fold increase relative to the highest reported value for Bacillus subtilis spore production Introduction Under conditions of extreme nutrient limitation, Bacillus subtilis undergoes a differentiation process that converts the rod-shaped bacterial cell into a dormant spore, highly resistant to physicochemical stresses (1) The spore is released at the end of the developmental process upon lysis of the mother cell; later, if appropriately stimulated, it can initiate germination, which leads again to vegetative growth (1, 2) Recent studies have demonstrated that spores of Bacillus spp (3-5) and in particular those of the nonpathogenic species B subtilis (6-8) may be successfully used as competitive exclusion agents The use of probiotics and competitive exclusion agents in animal husbandry has gained increased attention because of the rise of multiple antibiotic resistant bacterial pathogens, in association with the extensive use of antibiotics as growth promoters The use of spores as probiotics presents several advantages * To whom correspondence should be addressed Tel: + 351 21 446 94 80/3 Fax: + 351 21 446 93 90 E-mail: cunha@itqb.unl.pt † Instituto de Biologia Experimental e Tecnolo ´ gica ‡ Instituto de Tecnologia Quı´mica e Biolo ´ gica § Faculdade de Cie ˆ ncias e Tecnologia, Universidade Nova de Lisboa 10.1021/bp050062z CCC: $30.25 including the ease with which spores can be produced, their long storage life, and survival to the gastric barrier (4, 5, 9) In the agricultural industry spores are receiving increasing attention as potential alternatives to antibiotics as growth promoters (10) Probiotics and competitive exclusion agents are thought to enhance the gut microflora by preventing the colonization of the gastrointestinal tract by pathogenic bacteria (11, 12) There are four basic ways in which this might be achieved: (i) immune exclusion of pathogenic bacteria; (ii) exclusion of a pathogen by competitive adhesion, (iii) synthesis of antimicrobial substances that impair colonization of the gastrointestinal tract by pathogens, and (iv) depletion of or competition for essential nutrients (4-6, 13) Industrial exploitation of spores requires high cell density bioreaction and good sporulation efficiency At laboratory scale, sporulation is normally induced by growth and nutrient depletion in media such as Difco Sporulation Medium (DSM) The end of the exponential phase of growth is defined as the onset of sporulation, and the production of heat-resistant spores takes approximately h to be completed Under ideal conditions, the culture will initiate sporulation at a cell density of about 108 cells mL-1, and typical sporulation efficiencies will be in the range of 30-100% (14) One reasonable way © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 07/01/2005 Biotechnol Prog., 2005, Vol 21, No to increase spore production is to achieve high cell density cultivation and subsequently allow sporulation to occur (15, 16) Although fed-batch bioreactions have been frequently used to increase cell densities (17, 18), this technique has not been applied for B subtilis spore production The concentration of spores reported in the literature covers a wide range, depending mainly of the used strain: 1.0 × 105 spores mL-1 (19), 6.4 × 108 spores mL-1 (20), 1.0 × 109 and 2.0 × 109 spores mL-1 (21, 22 respectively); so far the highest reported value is 3.0 × 109 spores of B subtilis per mL-1 (23) In this work, the effect of the dissolved oxygen level, pH, and nutrient concentration on the extent of B subtilis growth and sporulation has been investigated in batch bioreactions A fed-batch bioprocess was developed, with the addition of a nutrient feeding solution The nutrient feed started before the complete depletion of the nutrients present in the media, at the middle of the exponential growth phase, thus before start of the sporulation process (24) The feeding solution was added using an exponential feeding profile obtained from the mass balance equations (17) The overall biomass balance equation was expressed in terms of specific glucose consumption, and the feeding profile was directly proportional to the glucose consumption Under controlled fed-batch conditions the maximum spore concentration achieved was 7.4 × 109 spores mL-1, corresponding to a 20-fold increase when compared to the results obtained with this strain under batch cultivation and being 2.5 times higher than the best results previously reported (23) Materials and Methods Strain The wild-type Spo+ B subtilis strain MB24 (trpC2 metC3) (25) was used for all the experiments A spore stock of this strain was prepared, divided in 1-mL aliquots and stored with 30% of glycerol in liquid nitrogen Culture Media Luria-Bertani (LB) medium was used for the measurements of vegetative cell and spore concentrations, its composition being yeast extract g L-1, peptone 10 g L-1, and NaCl 10 g L-1 Difco sporulation medium (DSM) [bacto nutrient broth g L-1, KCl g L-1, and MgSO4 0.25 g L-1 (14)], used for inocula preparation and batch and fed-batch cultures, was sterilized at 121 °C for 30 To L of this solution were added mL of each of the following filter-sterilized solutions: Ca(NO3)2 M, MnCl2 10 mM, and FeSO4 mM (14) The 2x-Difco sporulation medium (2 DSM) contains double strength of all the components of DSM Fed-batch bioreactions were performed using a solution with the following composition: Bacto nutrient broth 120 g L-1, KCl g L-1, MgSO4‚7H2O 7.7 g L-1, and glucose 52.5 g L-1, sterilized at 121 °C for 30 To L of this solution, 15 mL of each of the following filter sterilized solutions were added: Ca(NO3)2 M, MnCl2 10 mM, and FeSO4 mM Inocula Preparation A 100-mL Erlenmeyer flask containing 20 mL of DSM was inoculated with one cryovial of MB24 from the frozen stock The seeded culture was incubated at 37 °C and 150 rpm on a rotary shaker for 16 h to a final optical density of approximately 2.0 The cells were then used to inoculate the 2-L bioreactor at an inoculum size of 1% (v/v) Batch Bioprocess A 2-L bioreactor (Biostat B, B Braun, Germany) was inoculated with 20 mL of seed culture for a final volume of L Cultivation temperature 1027 and aeration rate were maintained constant at 37 °C and L min-1, respectively The dissolved oxygen concentration was maintained above the required value for each experiment by varying the agitation rate between 100 and 200 rpm When required, cultivation pH was controlled at the desired values for each experiment with the addition of NaOH N or H2SO4 N Whenever necessary an antifoaming agent (SAG-471, 0.5 g L-1) was automatically added to the bioreactor Fed-Batch Bioprocess A 2-L bioreactor (Biostat B, B Braun, Germany) was inoculated with 20 mL of seed culture Cultivation conditions were controlled as described for the batch bioprocess, and the experiment was initiated with 1.3 L of DSM containing 3.5 g L-1 of glucose The bioreaction was conducted in three stages: batch culture for the first h, fed-batch for the next h, and finally batch culture until the end of the run for a total of approximately 45 h At the middle of the exponential growth phase, the nutrient feeding was initiated Approximately 300 mL of the feeding solution was added using an exponential feeding profile obtained from the mass balance equations as earlier indicated The feeding rate profile was determined using simple mass balances based on a Monod-type kinetic model: X ) X0 eµ‚t (1) where X is the biomass concentration (g L-1), X0 is the biomass concentration at the beginning of the fed-batch phase (g L-1), µ is the specific growth rate (h-1), and t is the time length of the bioreaction Considering constant specific glucose consumption, a constant glucose concentration in the cultivation medium was achieved by feeding the concentrated glucose solution according to the following equation: Q ) Q0 eµ‚t (2) where Q is the feeding solution flowrate, and Q0 is the feeding solution flowrate at the beginning of the fed-batch phase (g h-1) Glucose feeding solution addition to the bioreactor was controlled using a weight control loop A balance was used to measure the addition flask weight, and a weight profile over time was defined using the following equation: ∆W ) ∫0tQ0 eµ‚t (3) where Q is the feeding solution flowrate (g h-1), W is the weight of the feeding solution (g), Q0 is the feeding solution flowrate at the beginning of the fed-batch phase (g h-1), µ is the specific growth rate (h-1), and t is the time length of the bioreaction (h) By analytical integration of the previous equation, the following formula was obtained and used to control the glucose feeding rate to the bioreactor: ∆W ) Q0 µ‚t (e - 1) µ (4) This fed-batch strategy was designed with two main objectives: avoid glucose limitation during the vegetative growth phase, as this would initiate sporulation during this stage of culture, and keep glucose concentration below 3.5 g L-1, as higher concentrations reduce spore production Biotechnol Prog., 2005, Vol 21, No 1028 The necessary kinetic parameters as Q0 ) 16 g h-1 and µ ) 1.04 h-1 were determined from batch experiments Optimization of these parameters was not performed as the used ones where able to fulfill the above glucose concentration criteria Glucose Determination One milliliter of culture medium was clarified by centrifugation at 14 000g for Glucose concentration in the supernatant was determined using a glucose dehydrogenase based kit as described by the manufacturer (Glucose HK, Sigma Diagnostics) Cell Growth Determination Optical density (OD) measurement at 595 nm was used for cell growth monitoring Whenever necessary samples were diluted to a final OD value lower than 0.5 Determination of Titers of Vegetative Cells and Spores Serial dilutions of the cell suspension to be tested were prepared and 10 µL of each dilution was inoculated to a 96-well plate containing 180 µL of LB media For each dilution 10 replicates were prepared Plates were incubated at 37 °C for 24 h and cell concentration was determined using the Reed and Muench method (26) Spores were counted using the same method, but the plates were heated to 80 °C for 20 before incubation Sporulation Efficiency and Spore Fraction Sporulation efficiency was defined as the percentage of the vegetative cells that undergo a complete sporulation process yielding heat-resistant spores and was calculated as the ratio between the final spore titer and the maximum of the vegetative cell titer reached during the bioreaction (14) The spore fraction was defined as the percentage of spores at a given time and was calculated as the ratio between spore concentration and total cell concentration (spores and vegetative cells) in each sample Results Effect of pH Under a noncontrolled pH bioreaction a high pH variation was observed: at the beginning of the exponential growth phase the pH decreased from 6.7 to 6.5, then a sharp increase to 8.1 was observed until the end of the exponential growth phase, and a slow increase to pH 9.0 occurred during the sporulation process In this experiment the maximum vegetative cell concentration achieved was 2.6 × 109 cells mL-1 but the sporulation efficiency was low (approximately 16%), leading to a final spore concentration of 4.2 × 108 spores mL-1 To determine the effect of the pH on B subtilis growth and sporulation, batch cultures at various pH were performed; the results depicted in Figure 1A and B show that when the pH was maintained at a constant value during the whole experiment, a significant increase of the sporulation efficiency was achieved This suggests that if the pH is kept constant a higher synchronizm of sporulation is achieved Within the pH range of 6.0-9.0, the sporulation efficiency did not depend of the pH value, being approximately constant at 50%, whereas a decrease in pH to 5.0 reduced the sporulation efficiency to 6% At pH 7.5 the spore fraction at the end of the run was slightly higher than in all other experiments, with an increase in the maximum vegetative cells concentration up to 7.5 × 109 cells mL-1 and a sporulation efficiency of approximately 50% This led to a final spore concentration of 3.6 × 109 spores mL-1, which corresponds to a 9-fold increase when compared to the batch performed without pH control Effect of Dissolved Oxygen Concentration To investigate the effect of dissolved oxygen concentration Figure Effect of pH on Bacillus subtilis growth and sporulation under batch cultivation A: (b) spore concentration (spores mL-1); (O) vegetative cell concentration (cells mL-1) B: ([) sporulation efficiency (%) and (0) spore fraction at the end of the run (%) on B subtilis growth and sporulation, a 2-L batch cultivation with dissolved oxygen concentration above 10%, 30%, and 50% of air saturation was carried out (Table 1) These results indicate that this parameter did not influence the microorganism growth, although a slightly higher spore concentration was reached controlling the dissolved oxygen concentration above 30% of air saturation Effect of DSM and Glucose Concentration The effect of DSM concentration was investigated in 2-L batch cultivations The results depicted in Table indicate that with the duplication of DSM concentration (2 DSM), although the maximum vegetative cell concentration reached approximately the same value, a significant increase of the sporulation efficiency was achieved from 48% to 77% This effect led to a 50% increase in the final spore concentration, reaching 4.8 × 109 spores mL-1 The effect of glucose on B subtilis growth and sporulation was also evaluated in 2-L batch bioreactions by varying the initial glucose concentration between 3.5 and 20 g L-1 (Figure 2A and B) The maximum vegetative cell concentration increased with the increase in glucose concentration up to g L-1, remaining constant for higher concentrations, as shown in Figure 3A However, a decrease in sporulation efficiency with the increase of glucose concentration was observed As shown in Figure up to g L-1 all the glucose initially added to the medium was consumed before the end of the exponential growth, while for higher initial glucose concentrations, there was still glucose consumption during the stationary phase Fed-Batch Bioreactions A fed-batch cultivation for B subtilis spore production was developed at 2-L bioreaction scale The cells were initially grown, in batch mode, in 1.3 L of DSM containing 3.5 g L-1 of glucose; then a nutrient feed was started at the middle of the exponential growth phase, before the complete depletion Biotechnol Prog., 2005, Vol 21, No 1029 Table Summary of the Batch and Fed-Batch Cultivations of Bacillus subtilisa PH sporulation glucose vegetative cells spores efficiency pO2 DSM concn -1 -1 -1 (%) (%) concn (g L ) (10 mL ) (10 mL ) ncc 5.0 6.0 6.5 7.0 7.5b 8.0 9.0 30 30 30 30 30 30 30 30 1x 1x 1x 1x 1x 1x 1x 1x Effect of pH (Batch) 2.6 1.0 4.5 4.8 6.9 7.5 6.1 1.7 0.4 0.1 2.2 2.3 3.5 3.6 2.8 1.0 15.3 10.0 48.8 47.9 50.7 48.0 45.9 58.8 7.5 7.5 7.5 10 30 50 1x 1x 1x Effect of pO2 (Batch) 6.1 2.8 6.6 3.5 6.8 3.2 45.2 53.2 46.4 7.5 7.5 30 30 1x 2x Effect of DSM (Batch) 7.5 3.6 6.2 4.8 48.0 77.0 7.5 7.5b 7.5 7.5 7.5 30 30 30 30 30 1x 1x 1x 1x 1x Effect of Glucose (Batch) 3.5 10.5 4.3 5.0 21.9 5.6 10 19.9 4.7 15 22.2 3.7 20 20.0 3.4 40.9 25.6 23.6 16.7 17.0 7.5b 30 1x 3.5 Fed-Batch 36.0 7.4 20.5 a Results obtained at the end of the bioreaction b Optimal results c Not controlled Figure Effect of initial glucose concentration on Bacillus subtilis growth and sporulation under batch cultivation A: (2) spore concentration (spores mL-1), (O) vegetative cell concentration (cells mL-1) B: ([) sporulation efficiency (%) the growth phase (Figure 4C) During this exponential growth phase the agitation rate was increased from 100 to approximately 1000 rpm to compensate for the oxygen consumption rate (Figure 4A) At the end of the fed-batch phase, glucose was completely depleted from the medium causing a spike in the dissolved oxygen concentration, indicating the onset of the sporulation process Although a higher cell lyses occurred at this stage when compared to the batch experiments, an increase of heat resistant spores concentration was achieved due to the higher cell growth Discussion Figure Effect of initial glucose concentration on Bacillus subtilis growth and sporulation under batch cultivation A: optical density B: glucose concentration (g L-1) Initial glucose concentration (g L-1): (b) 0, ([) 3.5, (2) 5, (4) 10, (]) 15, (O) 20 of the nutrients present in the media, thus before the beginning of the sporulation process This feeding strategy permitted to extend the exponential growth phase (10 h after inoculation), leading to a maximum vegetative cell concentration of 3.6 × 1010 cells mL-1 at the end of The optimization of the cultivation parameters (pH, dissolved oxygen concentration, and media composition) for B subtilis growth and sporulation was performed in controlled batch cultivations at 2-L scale The results indicate that the sporulation efficiency was almost independent of pH values within the range 6.0-9.0, better results being achieved at pH 7.5 The dissolved oxygen concentration within the studied range (10-50% of the oxygen saturation concentration) did not significantly influence the microorganism growth, although a slightly higher spore concentration was achieved when controlling the dissolved oxygen concentration above 30% of air saturation Recently, it was shown that B subtilis, previously thought to be a strict aerobe, could also grow anaerobically (27) However, under anaerobic conditions sporulation efficiency is highly reduced (28) The need for aerobic conditions for efficient sporulation is in agreement with our observation that the concentration of dissolved oxygen is important for efficient spore production, a value above 30% being optimal An increase in glucose concentration up to g L-1 led to an increase of the maximum vegetative cell and spore concentration, while initial glucose concentrations higher than g L-1 inhibited sporulation The complete glucose 1030 Biotechnol Prog., 2005, Vol 21, No increased spore production As nutrient depletion is the main stimulus for sporulation, it is very important to achieve glucose depletion at the end of the exponential growth phase This fed-batch process conduced to an increase in spore production up to 7.4 × 109 spores mL-1, which is 2.5 times higher than the highest earlier reported value for B subtilis spore production Recently, strains of B subtilis have been isolated from the gut of various animals and characterized in view of their potential application as probiotics (32, 33) Being indigenous to the gut, spores of these strains may result in better probiosis In preliminary experiments using new B subtilis strains isolated from the gut of healthy animals the sporulation efficiency was almost 100% (data not shown) Precise regulation of growth and sporulation parameters are of great importance for obtaining reproducible and homogeneous spore batches To fully understand the nutrient requirements for growth and sporulation, determination of the carbon source mass balance should be performed for both stages, growth and sporulation Development of a chemically defined media would allow the optimization of a fed-batch process where sporulation efficiency could also be increased by defining feeding profiles to cope with the nutrient requirements for the sporulation process The methodology herein described will likely be applicable to the high efficiency production of spores from these as well as other strains of B subtilis whenever high yields of spores are desirable Acknowledgment This work was supported by the European Commission project “Spore Probiotics: An Alternative to Antibiotics” (QLK-CT-2001-01729) References and Notes Figure Fed-batch culture of Bacillus subtilis A: Dissolved oxygen level (pO2) and agitation profiles B: (-b-) optical density at 595 nm, (/) glucose concentration (g L-1) C: (2) spore concentration (spores mL-1), (O) vegetative cell concentration (cells mL-1) depletion at the end of the vegetative cell growth phase made it possible to synchronize the sporulation by creating the optimal conditions for sporulation to occur (16) Spo0A, the product of the spo0A gene, is a response regulator activated by phosphorylation in response to several internal and external stimuli and is the master regulator for entry into sporulation (2, 29, 30) Phosphorylated Spo0A stimulates its own synthesis and hence entry into sporulation, by promoting switching of spo0A transcription from a promoter active during vegetative growth to a promoter active at the onset of sporulation (29) Promoter switching is sensitive to catabolite repression (reviewed in ref 29), and thus it may be that under our conditions, excess of glucose inhibits sporulation by repressing transcription of the spo0A gene A controlled bioprocess comprising an initial and final batch phases and an intermediate fed-batch operation was developed to accommodate the physiology of the bioreaction and sporulation activity This may be related to sporulation being controlled by catabolite repression (31), as glucose may be involved in induction inhibition of several enzymes at least partially responsible for sporulation The fed-batch strategy applied had two main objectives: avoid glucose limitation during the vegetative growth phase, as this would induce sporulation, and avoid also concentrations higher than 3.5 g L-1 to achieve (1) Errington, J Regulation of endospore formation in Bacillus subtilis Nat Rev Microbiol 2003, 1, 117-126 (2) Piggot, P J.; Losick, R Sporulation genes and intercompartmental regulation In Bacillus subtilis and Its Closest Relatives from Genes to Cells; Sonenshein, A L., Hoch, J A., Losick, R., Eds.; American Society for Microbiology: 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A L Control of sporulation initiation in Bacillus subtilis Curr Opin Microbiol 2000, 3, 561-566 (31) Chevanet, C.; Besson, F.; Michel, G Effect of various growth conditions on spore formation and Bacillomycin L production in Bacillus subtilis Can J Microbiol 1985, 32, 254-258 (32) Barbosa, T M.; Serra, C.; Henriques, A O Gut sporeformers In Bacterial Spores: Probiotics and Emerging Applications; Ricca, E., Henriques, A O., Cutting, S M., Eds.; Horizon Scientific Press: London, 2004; pp 78-101 (33) Barbosa, T M.; Serra, C.; La Ragione, R M.; Woodward, M J.; Henriques, A O Screening for Bacillus isolates in the broiler gastrointestinal tract Appl Environm Microbiol 2005, 71, 968-78 Accepted for publication June 6, 2005 BP050062Z ... Regulation of endospore formation in Bacillus subtilis Nat Rev Microbiol 2003, 1, 117-126 (2) Piggot, P J.; Losick, R Sporulation genes and intercompartmental regulation In Bacillus subtilis and Its Closest... (7) Mazza, P The use of Bacillus subtilis as an antidiarrhoeal microorganism Boll Chim Farm 1994, 133, 3-18 (8) La Ragione, R M.; Casula, G.; Cutting, S M.; Woodward, M J Bacillus subtilis spores... O.; Cutting, S M Characterization of Bacillus probiotics available for human use Appl Environ Microbiol 2004, 70, 2161-2171 (10) Casula, G.; Cutting, S M Bacillus Probiotics: Spore germination