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Background  Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria–bacteriophage co-cultures, are required.    Results  In the current study, we manipulated the inoculum concentrations of  Staphylococcus aureus  and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 10 8  to 2 × 10 9  CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.1) to delay the lysis of the host. Bacterial concentration of 2 × 10 8  CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 10 10  plaque forming unit (PFU)/mL). With this combination of phage–bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic  Staphylococcus  phage was 8.63 × 10 10  ± 9.71 × 10 9  PFU/mL (5.13-fold increase).    Conclusions  Therefore,  Staphylococcus  phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12934-021-01549-8.

microbial cell factories

Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology