Diversity of protease-producing bacteria in the Bohai Bay sediment and their extracellular enzymatic properties
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Abstract: Protease-producing bacteria play key roles in the degradation of organic nitrogen materials in marine sediments. However, their diversity, production of proteases and other extracellular enzymes, even in situ ecological functions remain largely unknown. In this study, we investigated the diversity of cultivable extracellular protease-producing bacteria in the sediments of the Bohai Bay. A total of 109 bacterial isolates were obtained from the sediments of 7 stations. The abundance of cultivable protease-producing bacteria was about 104 CFU/g of sediment in all the samples. Phylogenetic analysis based on 16S rRNA gene sequences classified all the isolates into 14 genera from phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria, with Pseudoalteromonas (63/109, 57.8%), Bacillus (9/109, 8.2%), Sulfitobacter (8/109, 7.3%) and Salegentibacter (6/109, 5.5%) as the dominant taxa. Enzymatic inhibition tests indicated that all the tested isolates produced serine and/or metalloprotease, with only a small proportion producing cysteine and/or aspartic proteases. Several extracellular enzyme activities, including alginase, lipase, amylase and cellulose, and nitrate reduction were also detected for strains with higher protease activities. According the results, the protease-producing bacteria could also be participate in many biogeochemical processes in marine sediments. Our study broadened understanding and knowledge on the potential ecological functions of protease-producing bacteria in marine sediments.
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Key words:
- protease-producing bacteria /
- diversity /
- inhibition test /
- multi-extracellular enzymes
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Figure 1. Map of the Bohai Bay in the Bohai Sea of China showing the sampling sites (●). The map was created using DIVA-GIS software (http//www.diva-gis.org), and the sampling sites were added according to GPS records.
Figure 2. Phylogenetic tree of the protease-producing bacteria isolated from the Bohai Bay based on 16S rRNA gene sequences. Taxa and GenBank accession numbers in boldface were generated in this study. The tree was constructed by neighbor-joining method using MEGA version 6.0. Only bootstrap values greater than 50% are presented in the nodes. The scale bar represents 2% nucleotide substitution.
Table 1. Characteristics of the sampling stations and the distribution of different genera in these stations
Properties Station BH07 BH12 BH17 BH18 BH20 BH22 CFD39 GPS 38.35°N, 118.75°E 38.55°N, 117.95°E 38.55°N, 118.95°E 38.75°N, 118.95°E 38.75°N, 118.55°E 38.75°N, 118.15°E 39.07°N, 118.53°E Characteristics for sediment samples Depth/m 16.14 9.66 22.65 25.57 23.78 17.27 12.19 Temperature/°C 24.97 27.11 20.36 19.32 23.27 25.47 26.14 pH 8.11 8.08 7.91 7.90 8.01 8.18 8.08 DO 4.02 4.17 2.20 2.28 3.50 5.11 4.86 Sal 30.30 30.02 30.31 30.45 30.51 30.20 30.63 OrgC/% 0.42 0.51 0.55 0.60 0.54 0.57 0.87 OrgN/% 0.06 0.08 0.09 0.08 0.09 0.10 0.11 C/N1) 7.00 6.38 6.11 7.5 6.00 5.70 7.91 Genera distribution Proteobacteria Pseudoalteromonas 3 8 15 6 16 7 8 Shewanella 3 1 Marinobacter 1 3 Sulfitobacter 4 4 Celeribacter 1 1 1 Albirhodobacter 1 1 Firmicutes Bacillus 7 1 1 Halobacillus 1 1 1 Fictibacillus 1 Actinobacteria Micrococcus 1 1 Citricoccus 1 Brachybacterium 1 Bacteroidetes Arenibacter 2 Salegentibacter 2 2 2 Total strain number (109) Diversity index Shannon–Wiener (H′) 1.58 1.22 1.66 1.01 0.34 0.94 0.64 Simpson (D) 0.72 0.61 0.71 0.61 0.20 0.48 0.34 Pielou (J) 0.81 0.76 0.76 0.92 0.50 0.68 0.58 Note: 1) C/N is the abbreviation of OrgC/OrgN. 
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Table 2. Summary of the inhibition test and the extracellular enzyme production analyses of the tested strains isolated from the Bohai Bay
Genera Strain Inhibition ratio1) (I)/% H/C ratio2) Nitrate PMSF O-P E-64 P-A Casein Gelatin Elastin Amylase Cellulase Alginase Tween 80 Pseudoalteromonas 70004 43.64 22.87 0 0 1.77 3.00 1.27 0 0 0 0 – 70006 60.97 0 3.35 0 2.78 2.92 1.40 4.00 2.18 3.26 6.31 + 70032 80.75 46.83 0 0 1.54 3.10 0 0 0 0 1.70 70038 16.89 0 0 0 2.33 4.50 0 0 0 0 1.61 – 70039 43.83 0 0 0 2.50 4.71 0 0 3.68 1.11 3.27 70048 48.41 16.53 0 0 2.78 5.00 0 0 0 0 4.52 + 70051 71.35 45.19 0 2.99 3.25 2.67 1.86 0 0 0 2.59 – 70053 76.43 29.47 0 0 2.25 5.00 0 0 0 1.49 5.40 – 70056 80.05 0 0 0 2.78 4.14 0 0 0 3.60 0 – 70305 46.37 0 0 0 3.42 1.40 2.21 1.59 0 1.20 3.17 – 70306 49.18 34.79 4.11 0 2.87 3.22 1.58 1.76 1.08 1.25 1.51 + 70307 81.10 44.84 0 0 2.94 2.56 1.92 3.23 0 1.33 3.36 – 70308 99.23 48.20 5.80 0 2.75 2.87 1.58 2.07 0 1.16 3.83 + 70320 52.13 5.81 0 0 2.31 1.46 1.55 1.49 1.22 1.22 1.51 – 70325 39.57 6.21 0 0 3.14 1.27 1.70 1.98 0 1.44 2.99 + 70326 81.42 34.55 0 0 3.19 1.14 2.10 2.19 0 1.96 1.70 – 70333 29.63 6.34 0 0 2.62 1.20 1.55 1.64 0 1.20 2.79 – 70339 90.13 31.28 4.48 4.48 2.00 0 1.50 1.61 4.11 1.23 3.46 + 70342 85.92 3.20 0 0 2.18 1.45 1.58 0 0 0 0 – 70344 70.04 53.84 7.04 0 2.38 1.42 1.25 1.34 0 1.28 2.96 – 70354 51.04 20.35 4.25 0 3.43 2.85 1.73 1.26 0 1.32 1.33 – 70356 68.40 10.60 0 7.72 3.18 1.57 2.09 1.75 0 1.31 1.19 – 70359 77.04 42.95 0 3.74 3.42 1.80 1.60 1.83 0 1.86 2.64 + 70361 50.74 6.17 3.60 0 3.00 2.36 1.77 1.35 0 2.12 2.83 – 70365 92.29 27.77 0 0 3.21 3.29 2.50 1.34 0 2.18 0 + 70369 57.37 34.39 0 0 2.44 2.08 1.50 1.76 1.41 2.37 1.18 – 70373 95.19 44.79 0 3.12 3.29 3.17 1.50 2.97 1.12 3.36 2.73 + 70374 76.82 37.19 0 0 3.13 2.27 1.36 2.36 0 6.55 2.78 – 70375 61.08 0 2.23 0 2.93 2.88 2.11 2.89 0 4.63 0 – 70376 15.39 5.92 0 0 3.00 2.75 1.33 0 0 2.16 0 – 70379 42.46 51.17 3.70 0 2.80 2.64 1.80 1.27 0 3.29 2.79 – 70380 91.52 51.24 1.28 4.57 2.82 3.15 1.64 0 1.59 2.10 0 – 70413 99.18 32.76 0 0 2.29 1.27 1.33 1.40 0 2.62 2.71 – Bacillus 70013 63.69 8.67 0 0 1.80 3.67 0 0 0 8.74 0 – 70035 9.42 0 0 0 1.40 6.50 0 0 0 0 0 + 70037 19.80 3.89 0 0 2.14 7.00 0 0 0 2.96 2.04 – 70049 92.58 44.74 0 3.09 4.80 5.42 1.32 0 0 0 0 – 70055 31.98 18.38 7.45 7.63 1.36 3.86 0 0 0 0 0 – Fictibacillus 70014 43.21 1.84 0 0 1.67 5.00 0 0 0 2.52 0 – Micrococcus 70025 68.60 24.30 7.83 0 4.00 5.17 0 0 0 0 0 – 70027 61.26 17.12 9.01 0 3.80 4.14 0 0 0 1.88 0 – Halobacillus 70074 38.71 0 0 0 3.57 3.33 2.29 0 0 2.95 0 – 70075 63.57 15.94 0 0 2.00 4.00 0 0 1.37 0 0 – Salegentibacter 70304 35.37 6.19 7.24 0 4.60 1.29 5.00 0 0 2.63 0 + 70390 84.24 0 0 0 8.33 7.83 5.40 0 0 3.41 0 + 70400 42.39 0 0 0 5.00 4.60 3.57 0 0 2.00 0 – 70401 46.26 8.49 0 0 6.75 6.00 5.00 0 0 2.62 0 + 70416 20.22 1.31 0 0 6.00 3.00 5.50 0 0 2.11 0 + Marinobacter 70434 41.70 37.85 0 0 2.09 0 0 3.54 0 5.27 2.40 + 70435 38.74 35.58 0 0 1.28 3.19 0 3.82 0 7.84 2.40 + Note: 1) Inhibition ratio (I, %) was calculated by using control activity minus the relative activity of a sample with an inhibitor and the activity of a sample without any inhibitor was taken as a control. 2) H/C ratio is the ratio of the hydrolytic zone diameter versus the colony diameter of a colony on the plate. PMSF, phenylmethylsulfonyl fluoride; OP, 1, 10-phenanthroline; P-A, pepstatin A.
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