Bacillus subtilis Endospores at High Purity and Recovery Yields: Optimization of Growth Conditions and Purification Method

Tavares, Milene B.; Souza, Renata D.; Luiz, Wilson Barros; Cavalcante, Rafael Ciro Marques; Casaroli, Caroline; Martins, Eduardo Gimenes; Ferreira, Rita de Cássia Café; Ferreira, Luís Carlos de Souza

doi:10.1007/s00284-012-0269-2

Cited by 53 publications

(29 citation statements)

References 12 publications

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“…These values do not surpass the spore cell density reached by Chen et al [9], who reported a concentration of 1.52 9 10 10 CFU/mL for the B. subtilis WHK-Z12 strain, but to our knowledge, the sporulation efficiency reached in this work (93.5 ± 4.0 %) is the highest reported to date for B. subtilis strains that are in the range of 30-80 % [11,12,14].…”

Section: Resultscontrasting

confidence: 49%

“…Under ideal conditions, typical cell densities and sporulation efficiencies for B. subtilis will be in the range of 1.00 9 10 8 -1.52 9 10 10 CFU/mL [9][10][11][12] and Electronic supplementary material The online version of this article (doi:10.1007/s00449-015-1428-1) contains supplementary material, which is available to authorized users. 30-80 %, respectively using serial dilution and plate count methods [11][12][13][14]. Other studies have reported higher sporulation efficiencies or higher spore cell densities for other Bacillus species different from B. subtilis [3,6,15,16], but to our knowledge obtaining high values of both variables has not been reported yet.…”

Section: Introductionmentioning

confidence: 98%

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Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production

Posada-Uribe

Romero-Tabarez

Villegas-Escobar

2015

Bioprocess Biosyst Eng

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Bacillus subtilis spores have important biotechnological applications; however, achieving both, high spore cell densities and sporulation efficiencies in fermentation, is poorly reported. In this study, medium components and culture conditions were optimized with different statistical methods to increase spore production of the plant growth promoting rhizobacteria B. subtilis EA-CB0575. Key medium components were determined with Plackett-Burman (PB) design, and the optimum concentration levels of two components (glucose, MgSO4·7H2O) were optimized with a full factorial and central composite design, achieving 1.37 × 10(9) CFU/mL of spore cell density and 93.5 % of sporulation efficiency in shake flask. The optimized medium was used to determine the effect of culture conditions on spore production at bioreactor level, finding that maintaining pH control did not affect significantly spore production, while the interaction of agitation and aeration rates had a significant effect on spore cell density. The overall optimization generated a 17.2-fold increase in spore cell density (8.78 × 10(9) CFU/mL) and 1.9-fold increase in sporulation efficiency (94.2 %) compared to that of PB design. These results indicate the potential of B. subtilis EA-CB0575 to produce both, high spore cell densities and sporulation efficiencies, with very low nutrient requirements and short incubation period which can represent savings of process production.

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Section: Resultscontrasting

confidence: 49%

Section: Introductionmentioning

confidence: 98%

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production

Posada-Uribe

Romero-Tabarez

Villegas-Escobar

2015

Bioprocess Biosyst Eng

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“…B. anthracis spores were prepared as described (Tavares et al ., ). Briefly, cells from sporulating cultures (50 ml) after 24 h of growth in DSM were harvested by centrifugation at 4°C for 10 min at 3,630 × g .…”

Section: Methodsmentioning

confidence: 97%

“…B. anthracis spores were prepared as described (Tavares et al, 2013). Briefly, cells from sporulating cultures (50 ml) after 24 h of growth in DSM were harvested by centrifugation at 48C for 10 min at 3,630 3 g. The bacterial pellets were washed once with 0.5 volume of distilled water and suspended in 10 ml of 10 mM Tris-HCl (pH 7.2) containing 50 lg/ml of lysozyme (Sigma-Aldrich).…”

Section: Sporulation Efficiency and Spore Preparationmentioning

confidence: 99%

Poly(3‐hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation

Sadykov

Ahn

Widhelm

et al. 2017

Molecular Microbiology

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Numerous bacteria accumulate poly(3-hydroxybutyrate) (PHB) as an intracellular reservoir of carbon and energy in response to imbalanced nutritional conditions. In Bacillus spp., where PHB biosynthesis precedes the formation of the dormant cell type called the spore (sporulation), the direct link between PHB accumulation and efficiency of sporulation was observed in multiple studies. Although the idea of PHB as an intracellular carbon and energy source fueling sporulation was proposed several decades ago, the mechanisms underlying PHB contribution to sporulation have not been defined. Here, we demonstrate that PHB deficiency impairs Bacillus anthracis sporulation through diminishing the energy status of the cells and by reducing carbon flux into the tricarboxylic acid (TCA) cycle and de novo lipid biosynthesis. Consequently, this metabolic imbalance decreased biosynthesis of the critical components required for spore integrity and resistance, such as dipicolinic acid (DPA) and the spore's inner membrane. Supplementation of the PHB deficient mutant with exogenous fatty acids overcame these sporulation defects, highlighting the importance of the TCA cycle and lipid biosynthesis during sporulation. Combined, the results of this work reveal the molecular mechanisms of PHB contribution to B. anthracis sporulation and provide valuable insight into the metabolic requirements for this developmental process in Bacillus species.

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“…B. subtilis WW02 is a widely used strain, which is a derivative from B. subtilis 1012, a reference laboratory strain with a sequenced genome and known physiological features [18], [19]. Spores were prepared from this strain, as previously described [20]. Briefly, sporulation of B. subtilis was induced following cultivation in F medium for 72 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

et al. 2014

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Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.

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Bacillus subtilis Endospores at High Purity and Recovery Yields: Optimization of Growth Conditions and Purification Method

Cited by 53 publications

References 12 publications

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production

Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production

Poly(3‐hydroxybutyrate) fuels the tricarboxylic acid cycle and de novo lipid biosynthesis during Bacillus anthracis sporulation

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

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