Diversity and ecological function of urease-producing bacteria in the cultivation environment of Gracilariopsis lemaneiformis

Urease-producing bacteria (UPB) provide inorganic nitrogen for primary producers by hydrolysis of urea. They play an important role in marine nitrogen cycle. However, there is still incomplete understanding of UPB and their ecological functions in the cultivation environment of red macroalage Gracilariopsis lemaneiformis. This study comprehensively analyzed the diversity of culturable UPB and explored their effects on urea uptake by G. lemaneiformis. Total 34 isolates belonging to four main bacterial phyla i.e. Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria were identified through 16S rRNA sequencing and were screened for UPB by urea agar chromogenic medium assay and ureC gene cloning. Our data revealed that only 8 strains were found containing urease. These all UPB exhibited different urease activities by Berthelot reaction colorimetry assay. Furthermore, UPB with highest urease activity was selected to use as co-culture with G. lemaneiformis to explore its role in terms of promotion or inhibition of nitrogen uptake by G. lemaneiformis. The results showed that the urea consumption in culture media and the total cellular nitrogen in G. lemaneiformis found increased significantly in the UPB-co culture group than control i.e. in the sterile group (p < 0.05). Similarity, isotopic assay revealed that {delta}15N contents of G. lemaneiformis was significant higher in the UPB-co culture than in the control where {delta}15N-urea was the only nitrogen source in the culture media, indicating the UPB helped G. lemaneiformis to absorb more nitrogen from urea. Moreover, the highest content of {delta}15N was found in G. lemaneiformis with epiphytic bacteria, as compared to sterilized (control) showing that epiphytic bacteria along with UPB have compound effects in helping G. lemaneiformis absorb more nitrogen in urea. Taken together, these results provide unique insight into the ecological role of UPB and suggest that urease from macroalgae environment-associated bacteria might be important player in the marine nitrogen cycling. ImportanceTo the best of our knowledge, this is the first study ever conducted to isolate the culturable UPB from the cultivation environment of G. lemaneiformis by urea agar chromogenic medium assay, and also evaluate the effects of UPB on urea utilization in G. lemaneiformis by stable isotopic tracer technique. This study provides a new insight into the mechanism of organic nitrogen uptake and utilization in G. lemaneiformis, and is of great significance for a better understanding of the ecological role of functional bacteria (e.g. urease-producing bacteria) in the marine nitrogen cycling.