Ladderane lipids synthesised by anammox bacteria hold significant potential for applications in jet fuel, drug delivery, and optoelectronics. Despite the widespread use of anammox bacteria in nitrogen removal from wastewater, the optimal conditions for maximising ladderane production remain unclear, limiting their broader application. To address this, we operated a fed-batch bioreactor with anammox bacteria, gradually adjusting the pH from 6.5 to 7.5 while regularly sampling for microbial community composition (Illumina sequencing), proteins, and ladderane lipids (UHPLC-HRMS). Our findings reveal that ladderane production positively correlates with rising pH increasing nearly fivefold as pH rose from 6.5 to 7.5, with a notable shift towards lipids containing two ladderane alkyl chains at higher pH. However, the conditions at an alkaline pH range also induced mild stress in anammox bacteria, as evidenced by our proteomic and microbial community data. Therefore, we propose maintaining a pH above 7.5 to enrich ladderane-rich anammox biomass but emphasise the need for gradual adaptation. This approach could optimise anammox installations for producing high-value ladderane lipids from wastewater.

University of Chemistry and Technology Prague Department of Biochemistry and Microbiology Technická 5 166 28 Prague Czech Republic

University of Chemistry and Technology Prague Department of Food Analysis and Nutrition Technická 5 166 28 Prague Czech Republic

University of Chemistry and Technology Prague Department of Mathematics Informatics and Cybernetics Technická 5 166 28 Prague Czech Republic

University of Chemistry and Technology Prague Department of Water Technology and Environmental Engineering Technická 5 166 28 Prague Czech Republic

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