Development of bioprocesses for the production of a biological indicator for sterilization processes from Bacillus atrophaeus spores

Abstract

Abstract: The genus Bacillus includes a great diversity of industrially important strains, including Bacillus atrophaeus (formerly Bacillus subtilis var. niger). This spore-forming bacterium has been established as industrial bacteria in the production of biological sterilization indicators, in studies of biodefense and astrobiology methods, and as potential adjuvants or vehicles for vaccines, among other applications. Two novels, cost-effective B. atrophaeus Sterilization Bioindicator Systems (BIS) with high quality and performance were developed from soybean molasses and glycerol and compared with commercial BIS. The BISs were composed of a recovery medium and dry-fermented. The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The first goal of the present work was to use fermented material as the final product in the BIS, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand and sugarcane bagasse) was assessed by determining B. atrophaeus sporulation during solid-state fermentation (SSF). Sand proved to be the best inert support, which enabled the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with the recovery medium. The BISs were developed and optimized using a sequential experimental design strategy. For soybean-based BIS, the optimum recovery medium contained soluble starch (1.0 g/L), soybean molasses (30.0 g/L), tryptone (40.0 g/L), and bromothymol blue (0.02 g/L) at pH 8.5. The SSF conditions of the bioreactor and environmental humidity had no significant impact on spore yield and dry-heat resistance. The only substrate mineral that showed a positive effect was Mn2+, allowing Mg2+, K+, and Ca2+ to be eliminated from the formulation. Validation of optimized medium indicated a D160°C = 6.8 ± 1.0 min (3.6 min more than the minimum) and a spore yield = 2.3 ± 0.5 × 109 CFU/g dry sand (10,000 × initial values). Cost reduction was of 23.9% and process cycle time was also reduced from 29 to 15 days. The study of the growth characteristics and the metabolic and enzymatic profiles confirmed that sporulation through SSF of B. atrophaeus occurs by biofilm formation and that this model of fermentation promotes important phenotypic changes in the spores. This study proposes a new concept regarding bacterial biofilm formation by SSF. For glycerol-based BIS, the proposed recovery medium enables the germination and outgrowth of heat-damaged spores, promoting a D160ºC value of 6.6 ? 0.1 min. B. atrophaeus spore production by SSF reached 2.3 ± 1.2 x 108 CFU/g dry matter. Sporulation kinetics results showed that only 5 days were sufficient for this process. Cost breakdowns were from 41.8% (quality control) up to 72.8% (feedstock). A performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with regulatory requirements. Microwave disinfection tests demonstrated that the developed BISs were more resistant than the control BIS. Additional studies are necessary to determine if this is a positive factor for microwave medical waste treatment monitoring or if it may cause a false process failure indication. These processes may be utilized for spore production aimed at other applications or for the cost-effective production of spores from other Bacillus species.