pH Coupled Citric Acid Feeding Strategy to Promote Spore Production of Bacillus licheniformis

The effects of citric acid addition coupled with pH on cell growth, metabolism, and spore yield of B. licheniformis were studied in a 30 L bioreactor, and the gene expression differences related to energy metabolism, oxidative stress effect, and amino acid transport at the log-phase were examined by qPCR. Results showed that: Citric acid supplement coupled with pH7.5 during 19.5~28 h was conducted, reaching a final content of 78 μmol/L in the medium. The feeding strategy prolonged the periods of cell growth and spore formation, and the peak cells (36 h) reached 4.2×10¹⁰ CFU/mL that was 42.9% higher than the control (without citric acid supplement). Meanwhile, spore yield (36 h) reached 4.1×10¹⁰ CFU/mL, increased by 215.4% compared with the control (24 h, 1.3×10¹⁰ CFU/mL). The qPCR assays showed that the expression levels of icd, zwf and cydA was 2.6, 4.1 and 1.9 times higher than that of the control, respectively. Meanwhile, brnQ, vpr, yvbW, katA, yqjM, ahpF, sodA and abrB were decreased by 88%, 18%, 65%, 89%, 10%, 90%, 41% and 60% compared with the control, respectively. The data suggested that citric acid supplementation enhanced the efficiencies of TCA cycle, HMP pathway and electron transport, weakened the oxidative stress effects on cells, and improved the phosphorylation level of Spo0A, thus promoting the sporulation efficiency. The results would provide theoretical supports for the industrial production of Bacillus fermentation efficiently.