Diversity of the protease-producing bacteria and their extracellular protease in the coastal mudflat of Jiaozhou Bay, China: in response to clam naturally growing and aquaculture

Abstract

The booming mudflat aquaculture poses an accumulation of organic matter and a certain environmental threat. Protease-producing bacteria are key players in regulating the nitrogen content in ecosystems. However, knowledge of the diversity of protease-producing bacteria in coastal mudflats is limited. This study investigated the bacterial diversity in the coastal mudflat, especially protease-producing bacteria and their extracellular proteases, by using culture-independent methods and culture-dependent methods. The clam aquaculture area exhibited a higher concentration of carbon, nitrogen, and phosphorus when compared with the non-clam area, and a lower richness and diversity of bacterial community when compared with the clam naturally growing area. The major classes in the coastal mud samples were Bacteroidia, Gammaproteobacteria, and Alphaproteobacteria. The Bacillus-like bacterial community was the dominant cultivated protease-producing group, accounting for 52.94% in the non-clam area, 30.77% in the clam naturally growing area, and 50% in the clam aquaculture area, respectively. Additionally, serine protease and metalloprotease were the principal extracellular protease of the isolated coastal bacteria. These findings shed light on the understanding of the microbes involved in organic nitrogen degradation in coastal mudflats and lays a foundation for the development of novel protease-producing bacterial agents for coastal mudflat purification.

Impact of Sea Rice Planting on Enzymatic Activity and Microbial Community of Coastal Soils: Focus on Proteinase

“…Lysobacter, Thiobacillus, Antarcticibacterium, Sphingomonas, Salinimicrobium, Pontibacter, Gemmatimonas, Ellin6067, RB41, Gillisia, Zeaxanthinibacter, MND1, Erythrobacter, and Intrasporangium were dominant in sea rice soils, with the sum of 34.01%. The potential proteolytic activity of the top 50 abundant genera of the bacterial community was shown (Table S2), among which Lysobacter, Pontibacter, Arthrobacter, Pseudomonas, Bacillus, Streptomyces, and Planococcus has been previously reported with the proteinase-producing strains [23][24][25][26][27][28][29][30][31], Arthrobacter, Pseudomonas, Bacillus, Streptomyces, Planococcus, and Nocardioides were isolated as proteinaseproducing bacteria by the subsequent culture-dependent methods in this study. The genera in red font were isolated as proteinase-producing bacteria by the subsequent culture-dependent methods or have been previously reported with the proteinase-producing strains.…”

“…The proteinase-producing activity of the isolated culturable bacteria was detected using previously estabilished methods [25]. The protein substrate plates consisted of 0.2% yeast extract, and 1.5% agar in artificial seawater, with different protein substrates including milk powder (1.0%), casein, or gelatin (0.5%), respectively.…”

“…The supernatant was diluted to the proper concentrations with Tris-HCl buffer (50 mM, pH 8.0), and then incubated with different inhibitors, PMSF, OP, E64, and Pepstatin A, at 4 • C for one hour, respectively. The residual activity of the proteinase was detected using the previously estabilished method [25]. The sample incubated with Tris-HCl buffer in the absence of any inhibitor serves as a negative control.…”