Investigations in ultrasound-assisted anticoagulant production by marine Bacillus subtilis ZHX

Anticoagulants are the main drugs for the prevention and treatment of thromboembolism. However, most of the present anticoagulants have shortcomings and novel anticoagulants are in great demand. Marine microorganisms are an important source of new drugs. Therefore, in this study, ultrasound was applied to enhance anticoagulant accumulation by marine Bacillus subtilis ZHX. Ultrasound parameters were optimized by single-factor experiments exploring the effects of ultrasound power, duration, duty cycle and the cell growth phases. The optimum conditions were exponential prophase (5 h) with 25 kHz frequency, 140 W power, and a 40% duty cycle for 5 min. The maximum anticoagulant activity (55.36 U/mL) was 1.73 times that of the control group, and the fermentation time was shortened by 3 h. Under optimal conditions, ultrasound increased the carbon utilization by Bacillus subtilis ZHX without significant changes in morphology, favoring cell growth and anticoagulant production. However, excessive ultrasound caused intracellular damage, which inhibited biomass accumulation, decreasing anticoagulant activity and even leading to cell rupture. This is the first report on the use of ultrasound to enhance anticoagulant production by Bacillus, and it provides useful information for scaling-up the process.