Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11 https://doi.org/10.1007/s13131-020-1589-x http://www.hyxb.org.cn E-mail: [email protected]

Diversity of protease-producing bacteria in the Bohai Bay 701.5

sediment and their extracellular enzymatic properties 684.5 1, 4† 1† 2 2 3 1 Zhenpeng Zhang , Chaoya Wu , Shuai Shao , Wei Liu , En-Tao Wang , Yan Li * 668.6 1 Key Laboratory of Coastal Biology and Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of 656.2 Sciences, Yantai 264003, China 646.2 2 Life Science College, Yantai University, Yantai 264005, China 634.0 3 Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico 621.8 City D.F. 11340, México 611.8 4 University of Chinese Academy of Sciences (UCAS), Beijing 100049, China 599.7

Received 2 May 2019; accepted 24 June 2019 584.6

© Chinese Society for Oceanography and Springer-Verlag GmbH Germany, part of Springer Nature 2020 565.1

Abstract 547.6

Protease-producing bacteria play key roles in the degradation of organic nitrogen materials in marine sediments.535.5 However, their diversity, production of proteases and other extracellular enzymes, even in situ ecological525.6 functions remain largely unknown. In this study, we investigated the diversity of cultivable extracellular protease-515.7 producing bacteria in the sediments of the Bohai Bay. A total of 109 bacterial isolates were obtained from the505.8 4 sediments of 7 stations. The abundance of cultivable protease-producing bacteria was about 10 CFU/g 495.9of sediment in all the samples. Phylogenetic analysis based on 16S rRNA gene sequences classified all the isolates486.0 into 14 genera from phyla Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria, with Pseudoalteromonas476.1 (63/109, 57.8%), Bacillus (9/109, 8.2%), Sulfitobacter (8/109, 7.3%) and Salegentibacter (6/109, 5.5%) as the466.2 dominant taxa. Enzymatic inhibition tests indicated that all the tested isolates produced serine and/or456.3 metalloprotease, with only a small proportion producing cysteine and/or aspartic proteases. Several extracellular446.4 enzyme activities, including alginase, lipase, amylase and cellulose, and nitrate reduction were also detected for436.5 strains with higher protease activities. According the results, the protease-producing bacteria could also be426.6 participate in many biogeochemical processes in marine sediments. Our study broadened understanding and416.7 knowledge on the potential ecological functions of protease-producing bacteria in marine sediments. 406.8

Key words: protease-producing bacteria, diversity, inhibition test, multi-extracellular enzymes 393.2

Citation: Zhang Zhenpeng, Wu Chaoya, Shao Shuai, Liu Wei, Wang En-Tao, Li Yan. 2020. Diversity of protease-producing bacteria in the378.0 Bohai Bay sediment and their extracellular enzymatic properties. Acta Oceanologica Sinica, 39(8): 1–11, doi: 10.1007/s13131-020-1589-x 368.1

344.0

1 Introduction 311.9 pecially in the sediment ecosystems. However, the diversity of311.7

Polymeric and particulate organic matters carry abundant or-300.8 these bacteria and their extracellular enzymes remain largely un-300.4

ganic nitrogen (OrgN), which are considered the main nitrogen289.6 known. 289.1

sources in marine sediment environments (Thamdrup and Dals-278.5 Protease producing bacteria are widely spread in marine sed-278.1

gaard, 2008). In the nitrogen cycle, the OrgN are usually decom-267.4 iments. For example, 30 out of the 98 strains isolated from South-267.0

posed into dissolved OrgN, followed by a series of ammonifica-256.3 ern Okinawa Trough possessed protease-producing abilities, dis-255.9

tion, nitrification and denitrification processes that are carried245.1 tributed in the genera Bacillus, Cobetia, Halomonas, Pseudomo-244.8

out by marine microorganisms (Hunter et al., 2006; Thamdrup234.0 nas, Psychrobacter, Myroides, Planococcus, Sporosarcina and233.7

and Dalsgaard, 2008). Proteins are the main building blocks of222.9 Wangia (Dang et al., 2009). By using selective media, diverse pro-222.6

organismal tissues, and protease producing bacteria are known211.8 tease producing bacteria were isolated from the sub-Antarctic211.5

as the main degraders of organic nitrogen in the marine environ-200.6 sediment (Olivera et al., 2007), the deep South China Sea sedi-200.4

ment (Zhang et al., 2015; Zhou et al., 2009). These bacterial189.5 ment (Zhou et al., 2009), coastal sediments of King George Is-189.3

groups secrete extracellular proteases that degrade proteins into178.4 land, Antractica (Zhou et al., 2013), enclosed the Jiaozhou Bay178.3

peptides and amino acids, which can be easily taken up by other167.3 (Zhang et al., 2015) and the Laizhou Bay (Li et al., 2017). In these167.2

bacteria for subsequent metabolism and processing (Zhao et al.,156.1 studies, the protease producing bacteria were classified under156.1

online145.0 first 145.0 2012). Therefore, protease-producing bacteria play important the genera Alcanivorax, Alteromonas, Burkholderia, Caulobacter,

roles in the biogeochemical cycles of marine environments, es-133.9 Celeribacter, Halomonas, Hyphomonas, Idiomarina, Marinobac-131.8133.9133.8 129.5

Foundation item: The National Natural Science Foundation of China under contract Nos 31600009 and 31800099; the Key Research118.3

and Development Program of Hebei Province under contract No. 19273802D; the STS Program of Chinese Academy of Sciences and107.3

Fujian Province under contract No. 2017T3019; the Yantai Science and Technology Project under contract No. 2018ZHGY074; the96.3

Joint Fund of Jilin Province and Chinese Academy of Sciences for High-tech Industrialization under contract No. 2019SYHZ0036; En-85.3

Tao Wang was supported by the projects under contract Nos SIP 20150597 and 20160883 authorized by IPN, Mexico. 74.3

*Corresponding author, E-mail: [email protected] 63.3 † These authors contributed equally to this work. 52.3 48.0 291.5 540.0 55.0 303.5 2 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11

ter, Microbulbifer, Photobacterium, Pseudoalteromonas, Pseudo-730.0 being dumped into it resulted to it becoming the most heavily ex-730.0 monas, Psychrobacter, Rheinheimera, Ruegeria, Shewanella,719.1 ploited and polluted marine areas in China. Therefore, the de-719.2 Sphingopyxis, Sulfitobacter and Vibrio of the phylum Proteobac-708.3 terioration of water quality accompanied by algal blooms, have708.4 teria; Bacillus, Exiguobacterium, Halobacillus, Jeotgalibacillus,697.4 occurred as manifestations of severe environmental problems in697.7 Oceanobacillus and Planococcus of Firmicutes; Aequorivita, Asin-686.6 this sea (Feng et al., 2011; Hu et al., 2010; Mu et al., 2017). With686.9 ibacterium, Flavobacterium, Formosa, Gillisia, Lacinutrix,675.7 the increasing problems on water pollution, the microbial com-676.1 Psychroserpens, Salegentibacter, Olleya and Zobellia of Bacteroid-664.9 munity in this aquatic ecosystem, especially the bacteria have665.4 etes; Arthrobacter, Janibacter, Leifsonia, Microbacterium, Micro-654.0 abilities to degrade organic materials, such as protein, alginate654.6 coccus, Nocardioides, Nocardiopsis and Streptomyces of Actin-643.2 and some pollutant chemical compound, are also expected to643.9 obacteria, dominated by Alteromonas, Bacillus, Flavobacterium,632.3 vary. However, the diversity of protease-producing bacteria in633.1 Lacinutrix, Photobacterium, Pseudoalteromonas and Vibrio. And621.5 this ecosystem and the characteristics of the proteases they pro-622.3 analyses of hydrolytic abilities towards different protein sub-610.6 duce remain little understood. Understanding of the proteases611.6 strates and inhibition tests indicated that all of these bacteria599.8 and other extracellular enzymes produced by these bacteria600.8 could decompose casein and/or gelatin and produce serine588.9 could not only facilitate the exploration of the other novel en-590.1 and/or metalloproteases (Li et al., 2017; Olivera et al., 2007;578.1 zyme-producing bacteria for biotechnological or industrial ap-579.3 Zhang et al., 2015; Zhou et al., 2009, 2013). 567.2 plications but would also help elucidate in situ biogeochemical568.5 Although the culture independent approaches has been ap-556.4 processes mediated by these microorganisms. Aiming to uncov-557.8 plied to detecting microbial resource reserves including the un-545.5 er the diversity of cultivable protease-producing bacterial com-547.0 cultivable taxa, isolation remains a necessary approach to obtain534.7 munity in the Bohai Bay, we isolated the proteolytic bacteria and536.3 novel microbes especially for screening specific functional isol-523.8 characterized their extracellular proteases using hydrolytic activ-525.5 ates with biotechnological application potentials (He et al., 2016;513.0 ity towards different protein substrates. We also tested factors514.7 Sfanos et al., 2005). For example, recent studies on protease-pro-502.1 that could inhibit their activity. Lastly, several other extracellular504.0 ducing bacteria using culturing method still obtained new groups491.3 enzymes production and nitrate reduction abilities were also493.2 482.5 of bacteria possessing higher protease producing abilities than480.4 tested. previous studies (Li et al., 2017). 469.6 The Bohai Bay, located in the northern coast of China, covers458.8 2 Materials and methods 460.4 2 a total area of 14 700 km , corresponding to 20% of the total area448.1 of the Bohai Sea. It is the largest semi-enclosed shallow water437.3 2.1 Ocean sediment collection and physicochemical character de-438.3 basin in the western region of the Bohai Sea. It is an important426.6 termination 427.3 spawning and traditional fishing grounds, and an aquaculture415.9 A total of 7 sediment samples were collected from the Bohai416.4 zone for economically important fish, shrimp and crab species405.1 Bay (GPS: 38.35°–39.07°N and 117.95°–118.95°E, Fig. 1 and Table 1)405.6 2 (Fu et al., 2016; Hu et al., 2010; Sun et al., 2011; Mu et al., 2017). In394.4 in April 2014 using a 0.05 m stainless steel Gray O’Hara box394.7 addition, the Bohai Bay costal area is one of the three most383.6 corner as previously described (Li et al., 2017). For each sampling383.8 densely populated and developed zones in China. Large volumes372.9 site, triplicate surface sediment soil samples (0–5 cm depth) were373.0 of sewage water containing heavy metal and organic pollutants362.1 collected using sterilized 60 mL syringes (without Luer end) and362.1

118° 119° 120° 121°E

CFD39 39° N BH22 BH20 Bohai Sea BH18

BH12 BH17

Bohai Bay BH07 38° online first

Laizhou Bay

0 350 700 1 400 km

78.8 73.3

Fig. 1. Map of the Bohai Bay in the Bohai Sea of China showing the sampling sites (●). The map was created using DIVA-GIS software60.0

(http//www.diva-gis.org), and the sampling sites were added according to GPS records. 49.0 291.5 540.0 55.0 303.5 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11 3

then transferred to airtight sterile plastic bags and stored at 4°C730.0 hydrolysis zones were detected. Colonies with different morpho-730.0

until analysis in the laboratory (Li et al., 2017; Zhou et al., 2009). 719.1 logical characteristics (e.g., colony color, size and surface poly-719.0

For physicochemical analyses, sediment samples were collec-708.1 saccharides) were selected and further purified by streaking re-708.0

ted with the same procedure and stored in sterilized plastic bags697.1 peatedly on the same plate until pure colonies were observed. All697.0

at –20°C and in the dark during the cruise and were transferred to686.1 pure cultures were preserved at –80°C in 20% (v/v) glycerol. 686.0 –80°C immediately after arrival at the laboratory. Sediment sur-675.1

face temperature and salinity were detected using Conductivity,664.1 2.3 Phylogenetic analyses 664.0

Temperature, and Depth (CTD) Data Acquisition System (MIRAI653.1 Each pure isolate was incubated in marine broth 2216 medi-653.0

MR99), and pH was determined in situ using a pH meter. Organ-642.1 um (Li et al., 2017) at 25°C with shaking of 200 r/min. Their bio-642.0

ic carbon (OrgC) and organic nitrogen (OrgN) concentrations631.1 masses were separately harvested by centrifugation and the gen-631.0

were quantified using a PE 2400 series II CHNS/O analyzer (Per-620.1 omic DNA was extracted using a TIANGEN genomic DNA extrac-620.0

kin Elmer, USA). 609.1 tion kit for bacteria (TIANGEN, China). Both DNA quality and609.0

quantity were estimated in Nano OD2000C (Thermo). The 16S598.0

2.2 Isolation of protease-producing bacteria from sediment587.1 rRNA gene of all the isolates were amplified and sequenced us-587.0

Ssamples 576.1 ing the universal primer pairs 27F/1492R at PCR conditions de-576.0

Protease-producing bacteria were isolated from sediment565.1 scribed in Engel et al. (2004). Samples were sent to Beijing565.1

samples with a selective medium following previous studies (Li et554.1 AuGCT DNA-SYN Biotechnology Co., Ltd for sequencing using554.1

al., 2017; Zhou et al., 2009). In brief, three samples from the same543.1 the Sanger method (Sanger et al., 1977). The quality of se-543.1

station were mixed together in equal weight and 1 g of the freshly532.1 quences were manually checked in BioEdit software version 7.0532.1 –6 mixed sediment sample was serially diluted to 10 with steril-521.1 and searched for their most similar sequences against the NCBI521.1

ized artificial sea water. Aliquots of 100 μL of each dilution510.1 GenBank using the BLASTn approach and also with EzTaxon510.1 –1 –6 (10 –10 ) were separately spread on the screening plates com-499.1 server (http://www.ezbiocloud.net/). Most similar sequences499.1

posed of 2 g yeast extract, 3 g casein, 5 g gelatin, 15 g agar powder488.1 were downloaded and a phylogenetic tree was reconstructed488.1

in 1 L artificial seawater at pH 7.0 (Zhou et al., 2009). All of the in-477.1 based on 16S rRNA gene sequences using the neighbor-joining477.1

oculated plates were incubated at 25°C until colonies with clear466.1 method (Saitou and Nei, 1987) with Kimura’s two-parameter466.1

Table 1. Characteristics of the sampling stations and the distribution of different genera in these stations 442.6 Station Properties BH07 BH12 BH17 BH18 BH20 BH22 CFD39 GPS 38.35°N, 38.55°N, 38.55°N, 38.75°N, 38.75°N, 38.75°N, 39.07°N, 118.75°E 117.95°E 118.95°E 118.95°E 118.55°E 118.15°E 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 Brachybacteriumonline1 first 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.

291.5 540.0 55.0 303.5 4 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11

model in MEGA version 6.0 (Tamura et al., 2013). The topology of730.0 2.6 Production of other extracellular enzymes and nitrate reduc-730.0

the phylogenetic tree was evaluated by bootstrap resampling718.8 tion test 719.0

method with 1 000 replicates (Felsenstein, 1985). All sequences707.6 The 50 representative strains, which produced enough extra-708.0

obtained in this study were deposited in GenBank database696.4 cellular protease to conduct inhibition tests were selected to de-697.0

(Table S1). 685.2 termine other extracellular enzymes produced. Activities of al-686.0

Due to highly conserved 16S rRNA gene sequences, which674.0 ginase, lipase, amylase and cellulase were analyzed as previously675.0

limits the resolution in delineating at the species level (Li et al.,662.8 described (Kitamikado et al., 1990; Hansen and Sørheim, 1991;664.0

2017), all isolates were only classified at the genera level. Alpha651.7 Yadav et al., 2015), and using H/C ratio (hydrolytic zone/colony,653.0

diversity of the community for each sample was calculated at the640.5 H/C) as a proxy for enzyme activity for each substrate. Enzyme642.0

genus level using three indices, namely, Shannon–Wiener index629.3 substrates were obtained from Sigma Company. Nitrate reduc-631.0 620.0 (H′) that shows the genera richness at a sampling site, the618.1 tion assay was performed according to Hansen and Sørheim 609.0 Simpson index (D) that explains the genera dominance, and the606.9 (1991), in brief, the tested isolates were inoculated for 5 d in li- 598.0 Pielou index (J) that indicates species evenness in a community595.7 quid media, which contained 0.5 g MgSO4, 0.5 g K2HPO4, 1 g 587.0 (Hill et al., 2003). The biodiversity indices were calculated using584.5 KNO3, 20 g sucrose, 0.5 g NaCl in 1 L of artificial sea water. Then 576.0 the Vegan package (version 1.17–4) performed in the R environ-573.4 the test was read by overlaying the wells with a mixture of sulph- anilic acid and α-naphthylamine. Positive isolates produced565.0 ment (version 3.3.2; http://www.r-project.org/) (R Core Team,562.2 bright red discoloration within 2–3 min, while negative isolates554.0 2014). 551.0 did not change the color and turned red color after addition of543.0

powdered Zn. 532.0 2.4 Inhibition ratio (%) of different inhibitors on the protease528.8

activity 517.6 3 Results 510.0 All protease-producing bacteria isolated in this study were506.4

then cultivated in the liquid screening medium (screening media495.2 3.1 Sampling site and sample characteristics 488.0 without agar, 100 mL), and were incubated at 25°C with shaking484.1 The water depths ranged from 9.66 m in Station BH12 to 25.57477.0 of 200 r/min for about 72 h. Then, the liquid cultures were centri-472.9 m in Station BH18 (Fig. 1 and Table 1). All sediment samples466.0 fuged at 12 000 r/min for 2 min to collect the supernatant, which461.7 showed slight alkalinity with pH varying from 7.90 in BH18 and455.0 were subsequently used to measure the protease activity (Chen et450.5 8.18 in BH22. The OrgC and of OrgN concentrations in the sedi-444.0 al., 2003). The inhibition tests were performed as described pre-439.3 ments ranged from 0.42% to 0.87% and 0.06% to 0.11%, respect-433.0 viously (Li et al., 2017). In brief, 1 mL of the supernatants were di-428.1 ively, with BH07 and CFD39 presenting the lowest and the422.0 luted with 50 mmol/L Tris-HCl containing 2.0% (w/v) casein (pH416.9 highest values for both OrgC and OrgN. Furthermore, the highest411.0 8.0), which was pre-incubated with 1.0 mmol/L phenylmethyl-405.8 C/N ratio (7.91) was also observed in Station CFD39 and the low-400.0 sulfonyl fluoride (PMSF, Sigma; serine protease inhibitor),394.6 est (5.70) in Station BH22. 389.0 1.0 mmol/L 1, 10-phenanthroline (OP, Sigma; metalloprotease383.4

inhibitor), 0.1 mmol/L M E-64 (Merk; cysteine protease inhibitor)372.2 3.2 Isolation and quantification of protease-producing bacteria 367.0 and 0.1 mmol/L pepstatin A (P-A, Sigma; aspartic protease inhib-361.0 Colonies with different colors, morphologies and sizes ap-356.0 itor) at 25°C for 20 min. After incubation, the reaction was349.8 –2 –4 peared on the screening plates inoculated with 10 –10 dilu-345.0 stopped by adding 2 mL of 0.4 mol/L trichloroacetic acid. Finally,338.6 tions after incubating at 25°C for 1–5 d, with the richness of about334.0 the supernatant was neutralized with 0.4 mol/L sodium carbon-327.5 4 10 CFU (colony forming unit) per gram of sediment soil samples.323.0 ate, incubated with Folin–Ciocalteu’s reagent solution (Sigma)316.3 Furthermore, nearly 80% of the colonies exhibited proteolytic312.0 and the protease activity was separately measured at 660 nm for305.1 zones. No obvious correlation between the richness of protease-301.0 each sample (Chen et al., 2003; Li et al., 2017; Zhang et al., 2015).293.9 producing bacteria and organic material was observed. Finally,290.0 One unit of enzyme activity was defined as the formation of282.7 109 proteolytic colonies were purified and selected for sub-279.0 1 μmol tyrosine in 1 min (Li et al., 2017). The protease activity of271.5 sequent analysis. 268.0 the sample without any inhibitor was used as the positive control.260.3

Then, the inhibition ratio (I, %) was calculated as I = (A – A ) ×249.2 c s 3.3 Diversity of the protease-producing bacteria Isolated from sed-246.0 100/A , where A was the activity in the positive control, and A 238.0 c c s iments 235.0 that of the sample (Li et al., 2017; Zhang et al., 2015; Zhou et al.,226.8 High quality nearly full-length 16S rRNA gene sequences224.0 215.6 2009). Finally, only 50 out of 109 isolates produced enough en- (> 1 400 bp) were generated from all 109 isolates. Phylogenetic213.0 204.4 zymes useful in determining the inhibition ratio. analysis classified them into 14 genera in the phyla Proteobac-202.0

teria, Firmicutes, Actinobacteria and Bacteroidetes (Fig. 2, Table 1191.0 182.2 2.5 Hydrolytic activities of extracellular enzymes against casein, and Table S2). 180.0

gelatin and elastin 171.0 Two isolates from Station BH17 belonged to Arenibacter and 6169.0

A basic medium containing 0.2% (w/v) yeast extract, 0.5%159.9 isolates from BH17, 18 and 20 isolates belonged to Salegentibac-158.0 (w/v) peptone, 1.5% (w/v) agar wasonline dissolved in artificial seawa-148.7 ter in the phylum of Bacteroidetes. first Two isolates from BH22 and147.0 ter at pH 8.0, supplemented with 0.5% (w/v) each of casein, gelat-137.5 BH 17 were affiliated with Citricoccus and Brachybacterium, re-136.0

in or elastin to prepare the plates (Zhou et al., 2009). All 109 isol-126.3 spectively and another two belonged to Micrococcus in the125.0

ates from sediment samples were inoculated with sterilized115.1 phylum Actinobacteria. The remaining isolates were classified114.0

toothpick on the media plates and incubated at 25°C for 3 d.103.9 into 9 genera in Phyla Proteobacteria (84/109, 77.06%) and Firmi-103.0

Then, the diameters of the colony and the hydrolyzed zone were92.7 cutes (11.93%), including Pseudoalteromonas, Shewanella and92.0

measured for each isolate, and a ratio of the hydrolytic zone dia-81.6 Marinobacter within the class Gammaproteobacteria; Sulfitobac-81.0

meter vs. the colony diameter (hydrolytic zone/colony, H/C) was70.4 ter, Celeribacter and Albirhodobacter within the class Alphapro-70.0

calculated as a proxy for enzyme activity against each substrate59.2 teobacteria in Proteobacteria; Bacillus, Halobacillus and Fictiba-59.0

(Li et al., 2017; Zhou et al., 2009). 48.0 cillus within the class Bacilli in Firmicutes. 48.0 291.5 540.0 55.0 303.5 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11 5

Pseudoalteromonas (63/109, 57.8%), Bacillus (9/109, 8.3%),730.0 tease-producing bacteria were isolated from BH17, which730.0

Sulfitobacter (8/109, 7.3%) and Salegentibacter (6/109, 5.5%) were718.8 showed higher diversity than those isolated from other sampling718.8

the dominant genera, while Fictibacillus, Citricoccus and Brachy-707.7 sites. In contrast, only two genera were identified in Station707.7

bacterium were represented only by a single isolate for each. The696.6 BH20, representing the least diverse community among the 7696.6

most abundant genus Pseudoalteromonas was isolated in all sta-685.5 sediment samples. 685.5

tions and dominated in nearly all stations except BH07, which674.4 Twenty-eight of the 63 Pseudoalteromonas isolates from Sta-674.4

was dominated by Bacillus. Furthermore, a total of 9 genera pro-663.3 tions BH17, BH18 and BH20 formed a distinct cluster (Branch 1)663.3

online first

67.8 62.3

Fig. 2. 49.0 291.5 540.0 55.0 303.5 6 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11

online first

119.5 114.0

Fig. 2. Phylogenetic tree of the protease-producing bacteria isolated from the Bohai Bay based on 16S rRNA gene sequences. Taxa100.6

and GenBank accession numbers in boldface were generated in this study. The tree was constructed by neighbor-joining method89.6

using MEGA version 6.0. Only bootstrap values greater than 50% are presented in the nodes. The scale bar represents 2% nucleotide78.6

substitution. 67.6

291.5 540.0 55.0 303.5 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11 7

in the constructed phylogenetic tree (Fig. 2, also see Table 1 and730.0 activities of all the 50 sub-selected isolates with varying levels730.0

Fig. S1) that shared 99.7%–100% of 16S rRNA gene similarities.719.0 from 9.42% to 99.23%, indicating that all of the isolates produced718.9

Most similar references included Pseudoalteromonas atlantica708.0 serine proteases in different proportions. Furthermore, the en-707.8 T T NBRC 103033 and Pseudoalteromonas hodoensis H7 , which697.0 zyme activities of 6 isolates were inhibited (>90%) by PMSF,696.7

were isolated from seaweeds in Japan (Akagawa-Matsushita et686.0 demonstrating that they mainly or only produced serine pro-685.6

al., 1993) and coastal sea water in Korea, respectively (Chi et al.,675.0 teases. The enzyme activities of 40 isolates were inhibited by OP,674.5

2014), all of which possess the ability to decompose algal poly-664.0 among which 23 isolates were efficiently inhibited (20.35%–663.4

saccharides. Meanwhile, 25 isolates from 6 stations formed653.0 53.84%) and 18 isolates were slightly inhibited (1.31%–18.48%),652.3

Branch 2 (Fig. 2 and Fig. S2) with 99.6%–100% similarities, clus-642.0 while no inhibitory effect on 10 isolates was observed, suggesting641.2 T tering closely with Pseudoalteromonas spiralis Te-2-2 and P.631.0 that most of the isolates produced metalloproteases. Meanwhile,630.1 T haloplanktis NBRC 102225 . A total of 5 isolates (70002, 70006,620.0 40 out of 50 (accounting 80%) isolates were inhibited by both619.0

70008, 70024 and 70053) were identical to Pseudoalteromonas609.0 PMSF and OP at different levels indicating that most isolates in607.9 T distincta KMM638 . Strains 70038 and 70039 sequences were598.0 this study produced both serine proteases and metalloproteases.596.8

highly similar (99.7%) with 70040, which in turn were587.0 There were only 14 isolates that were inhibited by E-64 and 8 isol-585.7 T 99.5%–99.8% similar to P. distincta KMM638 . On the other hand,576.0 ates by PA, but the inhibition ratios were less than 10%, illustrat-574.6

Isolates 70023 and 70056 were 99.9% similar with each other, and565.0 ing that these isolates produced very low amounts of cysteine or563.5 T closest to Pseudoalteromonas mariniglutinosa KMM 3635 554.0 aspartic protease. The results suggest that nearly all extracellular552.4

(98.8%–99.9%). Shewanella isolates (70058, 70059, 70060 and543.0 proteases produced by the tested bacteria belonged to serine541.3

70319) shared 99.9%–100% similarities to Shewanella algidipiscic-532.0 protease and/or metalloproteases. 530.2 T 521.0 ola NBRC 102032 , while 4 Marinobacter isolates (70052, 70061, The diversity of extracellular proteases produced by the 109518.9 510.0 70434 and 70435) shared 99.7%–100% similarities among them isolates from the Bohai Bay was also investigated through the hy-507.6 T and most similar to Marinobacter lipolyticus SM-19 499.0 drolytic abilities (H/C ratio) against different protein substrates496.3 (99.6%–99.9%). Strains 70046 and 70069 shared 99.9% similarit-488.0 (Table 2 and Table S3). Most of the isolates formed apparent hy-485.0 ies. Three Celeribacter strains 70047, 70067 and 70078 were most477.0 drolytic zones on the plates containing casein or gelatin, except473.7 T similar with Celeribacter baekdonensis L-6 , and the 8 Sulfitobac-466.0 70391, 70424 and 70432 that could not hydrolyze casein, and462.4 T ter were most related to Sulfitobacter pontiacus ChLG-10 with455.0 70335, 70339, 70417, 70433 and 70434 that were not capable of451.1 99.9% sequence similarities. Strains 70025 and 70027 shared444.0 hydrolyzing gelatin. The Salegentibacter isolates 70390, 70400,439.8 99.9% similarities and were most related to Micrococcus yunnan-433.0 70401 and 70416 showed high caseinolytic activity with H/C ratio428.5 T ensis YIM 65004 . Strain 70028 was closely related to Citricoccus422.0 greater than 5.0, with 70390 exhibiting the highest H/C ratio of417.2 T parietis 02-Je-010 with 99.0% sequence similarity. Strain 70026411.0 405.9 T 8.33. Further, Salegentibacter (70390 and 70401), Bacillus (70035, was most related to Brachybacterium alimentarium CNRZ 925 400.0 70037 and 70049), Fictibacillus (70014), Micrococcus (70025) and394.6 with 99.0% similarity. Isolate 70014 was closely related to Fictiba-389.0 T Pseudoalteromonas (70048, 70053) presented strong gelatinolytic383.3 cillus arsenicus Con a/3 with 98.9% sequence similarity. Isolates378.0 activity with H/C ratio greater than 5.0, but it was the Salegen-372.0 70057 and 70075 shared identical sequences and were most re-367.0 T tibacter isolate 70390 that had the highest H/C ratio of 7.83. Only360.7 lated to Halobacillus litoralis SL-4 with 99.9% similarities, while356.0 T 46 (42.2%) isolates exhibited ability to hydrolyze elastin, with the349.4 70074 was most related to Halobacillus faecis NBRC 103569 with345.0 Salegentibacter isolates 70304, 70390, 70401 and 70416 being the338.1 99.9% similarity. Nine Bacillus isolates (Fig. 2) showed high simil-334.0 most active, and the latter having the highest activity ratio of 5.50.326.8 arities (>99.0%) in 16S rRNA gene sequences with reference323.0 All the elastinolytic isolates also showed caseinolytic and gelat-315.5 strains for different defined species, including Bacillus hemi-312.0 T T inolytic activities except for 70339 that could not hydrolyze gelat-304.2 centroti JSM 076093 , Bacillus frigoritolerans DSM 8801 and Ba-301.0 T in. Salegentibacter isolates 70390 and 70401 could hydrolyze all292.9 cillus altitudinis 41KF2b etc.. The 70068 and 70414 shared 99.2%290.0 281.6 similarity and they were closely related to Arenibacter echinorum279.0 the three protein substrates with an H/C ratio greater than 5. Fur- T T 270.3 KMM 6032 (99.9%) and A. hampyeongensis HP12 (99.5%), re-268.0 thermore, varied hydrolytic abilities were observed among the

spectively. Isolates 70304, 70390, 70401, 70410 and 70416 shared257.0 isolates even for the isolates having identical 16S rRNA gene se-259.0

high similarity with 70301 (99.9% similarities), and all were246.0 quences. 247.7 T closely related to Salegentibacter salinarum ISL-4 (99.6% se-235.0

quence similarities). 224.0 3.5 Other extracellular enzyme production and nitrate reduction225.6

Shannon-Weiner (H′) index was highest in Station BH17213.0 test 214.5

(1.66), followed by Stations BH07 (1.58) and BH12 (1.22) (Table 1).202.0 All 50 isolates showing inhibition effects were further evalu-203.4

The lowest value (0.34) was detected in Station BH20 where only191.0 ated for their hydrolytic abilities (H/C ratio) against the sub-192.3

two genera were found. The highest Simpson index (D) value180.0 strates alginate sodium, Tween 80, soluble starch and cellulose.181.2

(0.72) was found in Station BH07, followed by Stations BH17169.0 Around 39 (78%) out of the 50 tested isolates produced alginase,170.1

(0.71), BH12 (0.61) and BH18 (0.61). The lowest value was ob-158.0 with 10 isolates exhibiting H/C ratio greater than 3.0, with Isol-159.0 served in Station BH20 (0.20). Theonline Pielou (J) value ranged from147.0 ates 70013, 70374, 70434 andfirst 70435 presenting H/C ratios greater147.9 0.50 in Station BH20 to 0.92 in Station BH18. 136.0 than 5.0. All Salegentibacter isolates degraded alginate sodium ef-136.8 ficiently. On the other hand, 29 isolates (58%) formed precipit-125.7

3.4 Diversity of the extracellular proteases produced by the bac-114.0 ates on the Tween 80 plates, where 8 isolates showed H/C ratios114.6

teria 103.0 greater than 3.0, and 70006 and 70053 had greater than 5.0.103.5

The diversity of proteases produced by the bacteria was in-92.0 Meanwhile, 24 isolates (48%) formed hydrolytic zones on soluble92.4

vestigated through inhibitor analyses and their hydrolytic abilit-81.0 starch plate, in which 70006, 70307, 70434 and 70435 presented81.3

ies against different types of protein substrates (Table 2 and Ta-70.0 H/C ratios greater than 3.0. Only 9 isolates (18%) possessed the70.2

ble S3). Out of the 109 isolates, only 50 produced enough pro-59.0 abilities to hydrolyze cellulose and Isolates 70039 and 70339 ex-59.1

teases for enzymatic inhibition analysis. PMSF inhibited the48.0 hibited ratios greater than 3.0. Thus, most tested isolates pro-48.0 291.5 540.0 55.0 303.5 8 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11

Table 2. Summary of the inhibition test and the extracellular enzyme production analyses of the tested strains isolated from the Bohai730.0

Bay 719.0 Inhibition ratio1) (I)/% H/C ratio2) Genera Strain 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 46.26online8.49 0 0 6.75 6.00 5.00 0 first0 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.

duced alginase, nearly half of them produced amylase and lipase,58.9 ates (32%) tested positive on nitrate reduction test, including 4 of58.9 and about 20% of the isolates produced cellulases. Lastly, 16 isol-48.0 the 5 tested Salegentibacter isolates. 48.0 291.5 540.0 55.0 303.5 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11 9

4 Discussion 730.0 difficult to degrade (Li et al., 2017). Interestingly, all Salegentibac-730.0

In the present study, sediment samples in the seven sampling719.0 ter isolates in this study exhibited the highest proteolytic activit-719.0

sites at the Bohai Bay had relatively lower C/N ratios (5.70 to 6.91,708.0 ies against all the three substrates (Table 2), which was in accord-708.0

with average 6.66) and richness of cultivable protease-producing697.0 ance with the previous study (Li et al., 2017). 697.0 4 bacteria (10 CFU/g of sediment) compared with the sediment686.0 Alginic acid is the major polysaccharide component of brown686.0

samples from the other two semi-enclosed bays, Jiaozhou Bay675.0 algae (Anastasakis et al., 2011) and macroalgae, such as Lamin-675.0

and Laizhou Bay, in China (Li et al., 2017; Zhang et al., 2015). The664.0 aria longissima, which is naturally widely distributed and also664.0

high evenness of the protease-producing bacteria in the sedi-653.0 cultured in the Bohai Bay (Zhang et al., 2007). Most of the tested653.0

ment samples indicates a relatively stable population size. No642.0 50 isolates produced alginase (Table 2), indicating that they642.0

significant correlations were observed between bacterial di-631.0 could be capable of degrading dead algae as their carbon sources631.0

versity or composition and OrgC, OrgN, and their ratios (OrgC/620.0 in the sediments. In marine environments, OrgN, usually in the620.0

OrgN), consistent with previous reports (Li et al., 2017; Zhang et609.0 form of proteins and fats, come from dead animals (Alasalvar et609.0

al., 2015; Zhou et al., 2009, 2013). Station CFD39 near the newly598.0 al., 2002). In addition, some algae accumulate large amounts of598.0

reclaimed area characterized by more anthropogenic disturb-587.0 algal oil (DemiRbas and DemiRbas, 2011). Thus, lipase is also im-587.0

ance, was the most polluted among the seven stations. Despite576.0 portant for survival of bacteria in marine sediments. In this study,576.0

this, the bacterial community structure in this station was not sig-565.0 more than one half of the tested isolates produced lipase, indicat-565.0

nificantly different from the other six stations. 554.0 ing that these bacteria could degrade fats that rich in the sedi-554.0

Bacterial diversity has been rarely investigated in the Bohai543.0 ments. Starch and cellulose meanwhile are mainly produced by543.0

Bay. A recent investigation based on culture-independent (RFLP532.0 terrestrial plants and some green algae (Itoh, 1990; Smant et al.,532.0

of 16S rRNA gene) method revealed the diverse communities in521.0 1998; Tsekos, 1999; Viola et al., 2001). The detection of amylase521.0

sediments of the Bohai Bay, belonging to at least six defined510.0 production in nearly half of the tested isolates demonstrate the510.0

phyla and other unclassified phyla with Proteobacteria as the499.0 possibility that the sediment bacteria utilize algal materials as499.0

dominant phylum and Gammaproteobacteria as the dominant488.0 their nutrient source. However, the small proportion (18%) of488.0

class (Sun et al., 2011). Similar observations were seen in our477.0 cellulolytic bacteria in the tested isolates in this study suggest477.0

study, where both Proteobacteria and Gammaproteobacteria466.0 that most of the proteolytic bacteria used materials other than466.0

dominated the phylum and class groupings of the communities,455.0 cellulose as their C-source. 455.0

respectively. Furthermore, the dominance of Pseudoalteromonas444.0 Protease-producing bacteria are known as the main decom-444.0

(63 isolates, 88.7% of the Gammaproteobacteria isolates) in all433.0 posers of organic nitrogen, converting them into peptides and433.0

the seven samples was consistent with the results of previous422.0 amino acids (Zhao et al., 2012). However, only a few studies have422.0

studies in the South China Sea and Laizhou Bay sediments (Li et411.0 reported their abilities to reduce nitrate. In this study, nitrate re-411.0

al., 2017; Zhou et al., 2009). Firmicutes was the second most400.0 duction was detected only in 16 out of the 50 tested isolates. Nev-400.0

abundant phylum, dominated by Bacillus (9 isolates, 8.3% of all389.0 ertheless, this could indicate their capacity for anaerobic respira-389.0

the isolates and 61.5% of the Firmicutes isolates), which again378.0 tion (Putz et al., 2018), which could be an adaptation to the sedi-378.0

was consistent with previous reports in the sediments of coastal367.0 ment environment, allowing them to be more efficient in miner-367.0

sub-Antarctic, Jiaozhou Bay and Laizhou Bay (Li et al., 2017;356.0 alization of organic matter. The high proportion of Salegentibac-356.0

Zhang et al., 2015; Zhou et al., 2009). The Bohai Bay is a tradition-345.0 ter that possessed proteolytic capacity suggests that they play im-345.0

al aquaculture zone and a spawning ground for many marine an-334.0 portant roles in the degradation of organic materials in the sedi-334.0

imals (Sun et al., 2011). Bacillus species are known to degrade323.0 ments. Specifically, all the five tested Salegentibacter isolates had323.0

wastes from aquafarming such that of shrimps, while Pseudoal-312.0 the highest proteases and alginase activities, with four of them312.0

teromonas are associated with marine animals such as fish301.0 showing nitrate reduction. The Pseudoalteromonas sp. 70006301.0

(Gamal et al., 2016; Pujalte et al., 2007). In addition, Bacillus and290.0 possessed the highest amylase and lipase activities, while290.0

Pseudoalteromonas strains have been suggested to influence the279.0 Pseudoalteromonas sp. 70339 had the highest cellulolytic activity.279.0

overall community structure through production of inhibitory/268.0 Our results indicate that proteolytic bacteria play multiple roles268.0

antibiotic secondary metabolites (Bowman, 2007; Mondol et al.,257.0 in organic matter degradation, and the specificity of their en-257.0

2013). Thus, the high proportions of Pseudoalteromonas and Ba-246.0 zyme activities against different substrates (Table 2 and Table S3)246.0

cillus isolates may be in accordance with their potential ecologic-235.0 allow effective degradation of diverse and complex organic ma-235.0

al functions and antibacterial capacities. These results show that224.0 terials, which make them functional exist in multiple marine eco-224.0

the main proteolytic bacterial functional groups were similar in213.0 niches in the sediments of the Bohai Bay. 213.0

the marine sediments of the South China Sea, sub-Antarctic, An-202.0 In conclusion, this study revealed the community structure of202.0

tractica, Jiaozhou Bay and Laizhou Bay (Olivera et al., 2007; Zhou191.0 the cultivable protease-producing bacteria in the sediments of191.0

et al., 2009; Zhang et al., 2015; Li et al., 2017), despite their geo-180.0 the Bohai Bay, and the major extracellular proteases and other180.0

graphic differences. However, compared with previous studies,169.0 hydrolytic enzymes they produce. Pseudoalteromonas, Bacillus,169.0

this was the first time that bacteria belonging to Albirhodobacter,158.0 Sulfitobacter and Salegentibacter were dominant taxa in the com-158.0 Fictibacillus, Citricoccus, Brachybacteriumonline and Arenibacter were147.0 munities, with all isolates capablefirst of producing serine and/or147.0 reported to possess proteolytic abilities. These results show that136.0 metallo-proteases. In addition, most bacteria effectively de-136.0

the marine sediments may be rich resource of novel enzymes for125.0 graded casein and/or gelatin, and only a small portion exhibited125.0

bioengineering. 114.0 elastinolytic ability. Albirhodobacter, Fictibacillus, Citricoccus,114.0

Similar to previous studies on proteolytic bacteria in the sedi-103.0 Brachybacterium and Arenibacter were first time to be reported103.0

ments (Li et al., 2017; Zhang et al., 2015; Zhou et al., 2009, 2013),92.0 for protease production. Interestingly, all Salegentibacter strains92.0

strains detected in this study mainly produced serine and/or81.0 exhibited the highest proteolytic activities for all the three pro-81.0

metallo-proteases. Furthermore, only a small portion of the isol-70.0 tein substrates casein, gelatin and elastin, and they may be can-70.0

ates exhibited elastin-degrading abilities (Table 2 and Table S3).59.0 didates for novel proteases. Through the detection of other extra-59.0

This could be due to the high molecular size of elastin, making it48.0 cellular enzyme activities, such as alginase, lipase, amylase and48.0 291.5 540.0 55.0 303.5 10 Zhang Zhenpeng et al. Acta Oceanol. Sin., 2020, Vol. 39, No. 8, P. 1–11

cellulase and nitrate reduction, we established cultures that730.0 hai Sea. FEMS Microbiology Letters, 363(10): fnw086, doi:730.5

strongly produce extracellular enzymes with biotechnological718.8 10.1093/femsle/fnw086 720.7 Hill T C J, Walsh K A, Harris J A, et al. 2003. Using ecological diversity711.0 application potentials. Furthermore, this study broadened un-707.7 measures with bacterial communities. FEMS Microbiology701.2 derstanding and knowledge on the potential ecological functions696.5 Ecology, 43(1): 1–11, doi: 10.1111/j.1574-6941.2003.tb01040.x 691.5 of protease-producing bacteria in marine sediments. 685.4 Hu Ningjing, Shi Xuefa, Liu Jihua, et al. 2010. Concentrations and681.7 possible sources of PAHs in sediments from Bohai Bay and ad-672.0 Acknowledgements 663.2 jacent shelf. Environmental Earth Sciences, 60(8): 1771–1782,662.2 We are grateful to Xiaoke Hu for helping us to revise the652.1 doi: 10.1007/s12665-009-0313-0 652.5

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507.4

Supplementary information: 478.2

Table S1. List of accession numbers. 463.4

Table S2. Distribution and taxonomy affiliation of protease producing bacteria. 450.1

Table S3. The H/C ratio of all the strains isolated in this study grown on the plates containing casein, gelatin, elastin respectively.436.8

Fig. S1. The maximun-likelihood phylogenetic tree of the strains in Branch 1 in Fig. 2 based on the 16S rRNA gene sequences. The tree425.8

was constructed using MEGA version 6.0. The scale bar represents 0.5% nucleotide substitution. 414.8

Fig. S2. The neighbor-joining phylogenetic tree of the strains in Branch 2 in Fig. 2 based on the 16S rRNA gene sequences. The tree was401.5

constructed using MEGA version 6.0. The scale bar represents 0.5% nucleotide substitution. 390.5

The supplementary information is available online at https://doi.org/10.1007/s13131-020-1589-x. The supplementary information is377.1

published as submitted, without typesetting or editing. 366.1

The responsibility for scientific accuracy and content remains entirely with the authors. 352.8

online first

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