Mechanical resistance of higher EPS contents in larger granules restricts anammox bacterial growth

C isotope labelling and qPCR assays, we confirmed that larger granules with higher EPS content exhibited the higher maximum nitrogen removal activity but much lower bacterial growth yield, resulting in a significantly lower maximum specific growth rate (-26.8%), compared to smaller granules. Metagenomic sequencing revealed that anammox species were identical in different granules, and actual EPS production yields were similar in 15-day incubation, ruling out the possibility that more energy was diverted to produce additional EPS in larger granules. Interestingly, the EPS mechanical strength was significantly greater in large granules, which reduced cell membrane fluidity and severely deformed bacterial cells. These mechanical constraints imposed by the dense EPS matrix limited anammox bacterial proliferation and reduced their growth yield. Using low-intensity ultrasound to loosen EPS structure improved the growth yield of anammox bacteria in large granules, while also enhancing nitrogen removal activity. These together contributed to a substantial increase in bacterial growth rate (+153.3%). The findings highlight that physical and mechanical resistance imposed by EPS plays a previously overlooked role in bacterial growth, and provide the basis for promoting anammox bacterial proliferation within granules.