East China Sea is one of the four sea areas in China, which possesses peculiar ecological environment and many kinds of living creatures, especially the microorganisms. We established the East China Sea microorganism library (during 2006–2010) for the first time, which stored about 30000 strains that covered most kinds of the species. In this paper, 395 pure strains of East China Sea microorganism library which belong to 33 different genera were mainly introduced.
Since the Scottish scientist Alexander Fleming discovered penicillin from the
China is a vast ocean country, which includes four sea areas, such as Bohai Sea, Yellow Sea, South China Sea, and East China Sea. Our country possesses coastline of 18,000 kilometers and the island coastline of 14,000 kilometers, and has the sovereignty and jurisdiction over the sea area of about 3 million square kilometers spanning the tropical zone, subtropical zone, temperate zone, and cold zone. Besides, China Sea possesses four kinds of marine ecosystems, including coastal wetland ecosystems, coral reef ecosystems, upwelling ecosystems, and deep sea ecosystems, which produce many kinds of microorganisms and have abundant resources. The rest three sea areas, except for East China Sea, have been systematically studied [
East China Sea mainly has the upwelling ecosystems and deep sea ecosystems. On one hand, upwelling marine ecosystem possesses higher biological diversity than other ecosystem because of the rich nutrients; on the other hand, some animals and microorganisms with specific structures mainly exist in the deep-sea ecosystem. Overall, the East China Sea has great potential for drug development because of its rich and unique microbial resources.
In order to discover the bioactive secondary metabolites of the microorganisms from the East China Sea, it is essential to do the large-scale survey of the microbial resources of East China Sea. In this paper, we mainly reported the marine microorganisms, their biological activities such as antimicrobial, antitumor, antioxidant activities, and some secondary metabolites of the strains with biological activities in the East China Sea.
The sea water and sea sediment samples were collected in 2006 at a depth of 30 m in the East China Sea (27°15′50′′ N120°45′22′′ E ~ 31°24′7′′ N122°40′48′′ E), and we totally obtained 100 sea sediment sample and 122 sea water sample, which were all stored in refrigerator at −80°C. Weigh 5 g sea sediment, add 45 mL sterile artificial water, shake and mix fully, and stand settlement. Sterile sea water and the supernatant of the sea sediment were progressively diluted with sterilized artificial seawater step by step into 10−1, 10−2, 10−3, 10−4, and 10−5. Take 0.2 mL every gradient diluent and spread them into different medium plates, incubate at 25°C for 7–14 days, and then pick the single colony according to the differences of the sample and the morphological of the strains. Streak plate method combined with the microscope observation is used during the separation process. All these pure strains were now conserved by freezing at −80°C and at 4°C in our laboratory.
According to the method described by Rainey et al. [
All strains were respectively inoculated in tubes containing 5 mL corresponding liquid medium and cultured on a rotary shaker (150 rpm) at 28°C for
The entire supernatants of cultured broth were prepared as described above. Cytotoxic activities of these supernatants were evaluated by MTT method [
Seed culture (50 mL) of all strains was inoculated into Erlenmeyer flask (2000 mL) containing the liquid medium (500 mL) and cultured on a rotary shaker (150 rpm) at 28°C for 5 days. The fermented whole broth was centrifugated at 12000 rpm for 5 min and separated from supernatant and thallus. The supernatant was extracted with ethyl acetate for 3 times, and the combined extracts were evaporated in vacuum at 30°C to dryness to yield the crude extract, which was dissolved at a concentration of 1 mg/mL in methanol for further analyses.
The DPPH assay was evaluated according to the method described by Hu and Kitts [
The ABTS assay was evaluated according to the method of Kajal and Paulraj [
The strains F81612, F201721, F8712, F321122, F121122, and M44 have been isolated from sea sediment and sea water of East China Sea, respectively. They were all isolated on F1 medium with incubation at 28°C. F1 Medium: Glucose (6 g), peptone (5 g), and yeast extract (1 g) were dissolved in artificial seawater (1 L). A statistical methodology, combining Plackett-Burman design (PBD) with response surface methodology (RSM), was applied to optimize the biological activity of the strains in the fermentation [
The 6 entire culture broths were all centrifugated at 12000 rpm for 15 minutes, respectively, got the supernatants. The each supernatant was extracted with ethyl acetate for 3 times. The combined extracts were evaporated in vacuum at 30°C to dryness to yield crude extract. Then, the each crude extract was subjected to Sephadex LH-20 and silica gel column, middle-pressure LC with the gradient elution, and chromatographed on preparative HPLC columns repeatedly, which yielded many secondary metabolites.
The 16S rRNA gene sequence has been determined for a large number of strains. GenBank, the largest databank of nucleotide sequences, has over 20 million deposited sequences, of which over 90,000 are of 16S rRNA gene. This means that there are many previously deposited sequences against which to compare the sequence of an unknown strain [
Taxonomy of 395 strains.
Genus | No. | Genus | No. | Genus | No. |
---|---|---|---|---|---|
8 | 3 | 4 | |||
30 | 7 | 6 | |||
2 | 1 | 10 | |||
5 | 42 | 36 | |||
9 | 2 | 3 | |||
2 | 4 | 3 | |||
5 | 1 | 4 | |||
6 | 7 | 5 | |||
69 | 12 | 2 | |||
5 | 2 | 10 | |||
62 | 8 | 20 |
45 strains of the tested pure isolated microorganisms exhibited antibacterial activities, among of which most strains belong to the
Strains with strongest antibacterial activities.
Strain no. | Genus | |||
---|---|---|---|---|
ZJ8112 | ++ | − | − | |
F121112 | + | − | − | |
F8712 | − | − | − | |
F321122 | − | − | − | |
201721 | ++ | − | + | |
XB16 | − | − | − | |
MZ0306A2 | + | + | ++ | |
MZ0208A2 | − | + | ++ | |
MZ0204A3 | − | + | + | |
P3001 | − | + | +++ | |
P4004A | − | + | + | |
P16006B | − | +++ | − | |
P3009B | − | +++ | − | |
P16011A | − | + | − | |
P16010C | − | ++ | − | |
P4004C | − | ++ | ++ | |
XF22−2 | − | − | ++ | |
P4003B | − | − | + | |
P12012B | − | − | ++ |
Note: “+++”means the diameters of the inhibition zone exceeding 15 mm; “++” means the diameters of the inhibition zone 10–15 mm; “+” means the diameter of the inhibition zone exceeding 6–10 mm; “−”means no antibacterial activities.
The tested pure isolated microorganisms exhibited good cytotoxicity activities to several cell lines, like HepG2, Hela, MCF, and SMMC 7721. It means not all the strains are susceptible to all the tested cell lines, which signifies the differences between the genus and the strains. 63 strains of the tested pure isolated microorganisms exhibited cytotoxicity, among of which most strains were sensitive to the HepG2 cell line and their inhibitory ratio mostly exceed 20% (Table
Strains with strongest cytotoxic activities of the 395 strains.
Strain no. | Genus | Sensitive tumor strain | Inhibitory ratio |
---|---|---|---|
F321121 | SMMC 7721 | 17.6% | |
P12002 | HeLa, MCF | 35%, 12% | |
F81612 | HeLa, SW1990 | 39%, 9.3% | |
F81611 | HeLa | 29.1% | |
P3001 | HepG2 | 28% | |
P3002 | HepG2, MCF | 27% | |
P16006A | HepG2 | 29% | |
P16006B | HepG2 | 32% | |
P16001 | HeLa, MCF | 18.4%, 20.4% | |
P4003B | HepG2, P388 | 31%, 7.9% | |
P12006A2 | HepG2 | 31% | |
P4006B | HepG2, P388 | 42%, 13% | |
MZ0206A2 | SMMC7721, S180 | 11%, 19% | |
MZ0208A2 | SMMC 7721 | 21% | |
P16003 | HepG2 | 23% | |
P4004B | HepG2 | 31% | |
P12002 | HepG2 | 28.7% | |
P12012B | HepG2, SW1990 | 14.9%, 17% | |
P12004D | HepG2, S180 | 16.7%, 23% |
All the cultured broths of the strains were tested at a fixed concentration 500
Most strains of
Strains of
The antioxidant activity of the pure cultured strains was determined by the method of spectrophotometry DPPH
24 strains with best antioxidant activity in DPPH
30 strains with best antioxidant activity in ABTS
In our screening program for bioactive principles from marine microorganisms, we pick up 6 strains (F81612, F201721, F8712, F321122, F121122, and M44) of different genus with strong biological activities to study their bioactive secondary metabolites (Table
Overview of the 6 strains with biological activities.
No. | Genus | Biological activity | Main type of the secondary metabolites |
---|---|---|---|
F81612 | Antibacterial | cyclopeptide, indole derivatives, and macrolide | |
F201721 | Antibacterial | cyclopeptide, indole derivatives, and macrolide | |
F8712 | Cytotoxicity | cyclopeptide | |
F321122 | Cytotoxicity | cyclopeptide and indole derivatives | |
F121122 | Cytotoxicity | cyclopeptide | |
M44 | Cytotoxicity | cyclopeptide |
The new secondary metabolites and the compounds with the good biological activities isolated from the strain F201721, F81612, F8712, F321122, F121122, and M44.
From the secondary metabolites of our finding, another important kind of the metabolites is macrolide. In 2007, our group found a new macrolactin named macrolactin Q which was isolated from a marine
According to the different sampling points and the different characteristics (morphous, sizes, and colours) of the pure strains, we totally obtained about 30000 pure strains, of which 52% strains belong to the actinomycete, 15% belong to the fungus, and 33% are bacteria. Firstly, we picked up 416 isolated bacteria according to their different morphologic and cultural characteristics to further study their taxonomy, biological activities, and secondary metabolites.
Based on the morphologic and cultural characteristics of the strains, firstly, it is known that majority of them belong to the Gram-negative bacteria. Secondly, more than 85% strains must be cultured in the medium with different concentrations of NaCl, the concentrations of NaCl which range from 2.5% to 15%. Thirdly, it is shown that these strains only need simple nutritional requirements, most of which possess extracellular enzymes and do not use sugar as carbon source and so on. Besides, 395 strains can be determined their taxonomic status, which belong to 33 different genera, and
In the recent years, there are many studies about the biological activities of the secondary metabolites isolated from the marine microorganisms, and these studies mainly focus on the antibacterial, cytotoxic, antioxidation, and antiviral, immunosuppressant activities [
On the large-scale activity screening of the 395 strains, 100 strains possess different biological activities, of which 45 strains have antibacterial activities, 63 strains have cytotoxicity activities, and 24 strains have antioxidant activities. By this screening, we found that the microorganisms of East China Sea have a wide range of biological activities, including antibacterial activities, cytotoxicity activities, and antioxidant activities, which possess the good potential for the development and utilization. It is good for us to further develop the medicinal microbial use of East China Sea on the foundation of the biological activity screening of the isolated pure strains.
In our screening program for bioactive principles from marine microorganisms, diketopiperazine and macrolide are the two important bioactive secondary metabolites. Diketopiperazines (DKPs), the smallest cyclic peptides, have been isolated from marine microorganisms (bacteria, fungi, and actinomycete) [
Besides, DKPs represent an important class of biologically active natural products, which play an important ecological role in antifouling [
Macrolactins are a group of 24-membered lactones with potent antibacterial or other activities, most of which were derived from the second metabolites of the marine microorganisms, while several of them were also produced by some soil microorganisms as well. There were total 22 isolated macrolactins reported, since the first one was isolated in 1989. Our group found a new macrolactin named macrolactin Q, which possesses strong antibacterial activity against
The East China Sea marine microorganism library (during 2006–2010) was established for the first time, which stored about 30000 strains that covered most kinds of the species. In this paper, 395 strains were mainly described, which belong to 33 different genera.
The authors declare that there is no conflict of interests.
Diphenyl-1-picrylhydrazyl
2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)
Diketopiperazines
Minimal inhibitory concentration.
This work was supported by National Hi-tech R&D Program to B. Jiao (no. 2007AA091501), X. Liu (no. 2006AA09Z416), and Q. Xu (no. 2006AA09Z425), Science and Technology Commission of Shanghai Municipality Program to B. Jiao (no. 09431901900).