Co-cultivation enhanced microbial protein production based on autotrophic nitrogen-fixing hydrogen-oxidizing bacteria

• Co-cultivation of N 2 -fixing HOB with other bacteria enhanced protein production. • The coculture produced protein superior to that of mixed communities. • Mutual benefits in the coculture probably enhanced protein synthesis. • The coculture had a low risk associated to bacterial toxins. • The highly integrated process could help to fix CO 2 and store renewable energy. Autotrophic N 2 -fixing H 2 -oxidizing communities produced sustainable microbial protein as an alternative to food and feed based on the Haber-Bosch process. Their dynamics and complexity make it challenging to control the composition and evaluate the product safety. Based on the isolates from a previously enriched community and their metabolism, this research studied the potential of binary cocultures for the production of safe protein. Compared to the monocultures of the isolates from Xanthobacter variabilis , i.e. the only N 2 -fixing H 2 -oxidizing species in the study, 31% of the 72 cocultures containing X. variabilis and the other isolates were promoted regarding growth due to co-cultivation, while 6% and 63% of the cocultures were inhibited and slightly affected, respectively. For the coculture where the cell count of X. variabilis was improved by 11.4 ± 3.5 times, its growth surpassed that of all monocultures and was high among the cocultures. Besides, the protein content, essential amino acid contents, and biomass yield of this coculture were 24 ± 2%, 28 ± 11% to 1.3 ± 0.3 times, and 26 ± 3% higher, respectively, than those of its X. variabilis monoculture. Compared to the mixed communities, the coculture had a similar biomass yield but superior protein quality. With the low predicted risk from bacterial toxins, the coculture had the potential for safe use and is currently the most suitable protein producer based on N 2 -fixing H 2 -oxidizing bacteria. In addition, the coculture independent of centrally synthesized Haber-Bosch nitrogen and its transportation had higher CO 2 fixation potential than H 2 -oxidizing bacteria grown on Haber-Bosch nitrogen producing CO 2 , converted solar energy more efficiently than agriculture, and can benefit in situ storage of solar and wind power.