A two-phase protocol for ambient hydrogen production using .

H production from green-microalgae, for energy purposes, is the ultimate goal of large-scale production. Here, we present a two-phase protocol for hydrogen production assay under ambient conditions using , which eliminates steps used previously, including centrifugation and resuspension with sulfur-deprived media. We detail steps for culture, acetate supply replenishment, anaerobic induction, and H quantification. This protocol enables large-scale experiments in an easy and cost-effective method while maintaining cells vital, crucial factors for transition to industrial scales. For complete details on the use and execution of this protocol, please refer to Elman et al. (2022).