Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity.

OBJECTIVES: In order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. METHODS: C. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response. RESULTS: In the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin. CONCLUSIONS: The best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response.

Optimization and scale-up of Clostridium perfringens type D culture and epsilon-toxin production: Effects of stirring, glucose and pH adjustment.

The effects of some main bacteria culture parameters including mixing rate, glucose (GC) concentration, steps of GC addition, and steps of pH adjustment on both C. perfringens bacteria growth and its epsilon toxin production in a bench-scale 20-L glass carboy were investigated. The optimized mixing rate of 300 rpm, GC concentration of 4 g L-1, and 3-step addition of GC resulted in the bacteria and toxin concentrations of 0.16 g L-1 and 330 ng mL-1, respectively. Also, the induction of a pH shock at the reaction time of 180 min led to the remarkable enhancement of toxin production (367 ng mL-1). Upon applying both optimized conditions for GC addition and pH adjustment, the high toxin concentration of 433 ng mL-1 was obtained. Using the constant mixing rate technique, the process was scaled up to a 1500-L industrial bioreactor, where its performance was close to the bench-scale bioreactor (i.e., toxin concentration of 419 ng mL-1). The results revealed the reliability of this method to economically improve and scale up the bacteria culture process, which can be further used for other microbial fermentations.