Establishing a reference cultivation protocol for systematic characterisation of the

Introduction: Halomonas species are increasingly recognised as promising next-generation industrial biotechnology chassis due to their capacity to grow under high-salt, non-sterile conditions, reducing contamination risk and eliminating the need for resource-intensive sterilisation. Despite this potential, most Halomonas species remain poorly characterised beyond initial isolation, limiting their adoption. Developing reference cultivation conditions for Halomonas genus, which could be employed as standard practice, is a prerequisite for extended biotechnological and industrial uptake. Methods: The maximum OD600 and maximum specific growth rates of 82 Halomonas strains representing 80 species were determined under their respective culture collection-recommended conditions, encompassing 27 unique sets of cultivation parameters. Results and Discussion: A subsequently developed reference cultivation protocol supported growth of 95% of strains in Luria-Bertani (LB) broth at either low- (2% NaCl; LB20) or moderate-salt (8% NaCl; LB80) concentrations at either 28 °C or 37 °C. Of the remaining 78 strains, only two performed significantly worse in both specific growth yield and maximum OD600 when a reference protocol was followed. Twenty-three strains were tested for growth at either 28 °C or 37 °C due to the differing recommended temperatures by the strain providers. Transition to salt-supplemented LB broth significantly increased specific growth rates in ten strains and maximum OD600 in eleven strains. Of the 82 strains, 67 were capable of growth in moderate-salt LB broth compared to only 29 in low-salt LB broth, demonstrating that elevated salinity is broadly permissive across the genus. With the exception of four strains, all members of the in-house collection were able to grow when a reference protocol was followed. The proposed reference culture conditions were LB80 at 28 °C for 35 strains, LB80 at 37 °C for 14 strains, either LB80 or LB20 at 28 °C for 13 strains, LB20 at 28 °C for ten strains, either LB80 or LB20 at 37 °C for five strains, and LB20 at 37 °C for one strain. This cultivation framework, comprising LB medium supplemented with 2% or 8% NaCl at 28 °C or 37 °C, is broadly applicable across the Halomonas genus and provides a standardised, resource-efficient foundation for systematic strain characterisation, comparative physiological studies, and future engineering efforts aimed at expanding the industrial utility of this promising microbial chassis.