Floating macrophyte phyllosphere as a habitat for methanogens

Methane generation has a significant impact on climate change. Aquatic plants have been widely studied for their role in methane emissions. Most research has focused on the contribution of the plant itself whereas the potential involvement of the phyllosphere microbiota associated with aquatic plants in methanogenesis remains largely unexplored. This study investigated the phyllosphere microbiota on both the top and bottom sides of lotus (Nelumbo nucifera), a commonly found aquatic plant in Asia, sampled from two urban ponds, using sequencing for both the 16S rRNA gene and the methanogenesis functional marker gene. Methanogenesis potential was assessed through laboratory-based 13C labeling experiments. Results show that methanogenic archaea were successfully detected on the floating lotus leaves but not on the standing leaves. Among the methanogens detected, Methanobacterium was identified as the predominant group, with significantly higher relative abundance on the bottom sides of floating leaves compared to the surrounding pond water. This study demonstrates the metabolic activity of the phyllosphere methanogens of floating lotus. The microbiota associated with the bottom sides of floating leaves exhibited the ability to produce methane and incorporate 13C carbon into methane during incubation. Overall, this study provides the first compelling genetic and functional evidence for the existence of epiphytic methanogens on the phyllosphere of aquatic plants. Hence, the phyllosphere of floating-leaved macrophytes represents a previously unrecognized habitat for methanogens and a potential microbial source of methane at the water-atmosphere interface.