The sensitivities of the cyanobacterium and associated bacteria to antibiotics
Antibiotics are commonly used to eliminate heterotrophic bacteria from cyanobacterial cultures [20,25,32]. In order to eliminate the heterotrophic contaminants, the sensitivities of the cyanobacterium and associated bacteria to antibiotics are investigated. Table 1 indicate that tetracycline, cephalosporin and streptomycin have obvious inhibiting effect on strain B905-1 with the antibacterial circle of 3.3, 2.4 and 1.8 cm, respectively, while kanamycin and penicillin have a negative inhibiting effect. In addition, the inhibition efficiency of tetracycline, cephalosporin, kanamycin, penicillin and streptomycin on the growth of M. aeruginosa FACHB-905 is 94.3 ± 3.5%, 88.5 ± 4.1%, 50.7 ± 2.4%, 5.6 ± 0.2% and 81.2 ± 3.9%, respectively (Fig. 2). It is demonstrated that M. aeruginosa FACHB-905 is significantly inhibited by all of the tested antibiotics except penicillins.
Table 1 Effects of antibiotics on cyanobacterium-associated bacterium and cyanobacterium
Antibiotics
|
Tetracycline
|
Cephalosporins
|
Kanamycin
|
Penicillins
|
Streptomycin
|
Antibacterial circle
|
3.3 cm
|
2.4 cm
|
—
|
—
|
1.8 cm
|
Inhibition efficiency
|
94.3 ± 3.5%
|
88.5 ± 4.1%
|
50.7 ± 2.4%
|
5.6 ± 0.2%
|
81.2 ± 3.9%
|
* and ** represent a statistically significant difference of p < 0.05 and p < 0.01 when compared to the control.
Fig. 2 The effects of antibiotics on the growth of M. aeruginosa FACHB-905. * and ** represent a statistically significant difference of p < 0.05 and p < 0.01 when compared to the control.
Effects of lysozyme on the cyanobacterium and associated bacteria
To obtain the axenic culture, the effects of lysozyme on the cyanobacterium and associated bacteria are investigated. The results demonstrate that both M. aeruginosa FACHB-905 and associated bacteria are reduced with the increasing of lysozyme concentration; moreover, the reduction of M. aeruginosa FACHB-905 is much more obvious (Table 2). As the lysozyme concentration increases from 1.0 to 10.0 mg mL-1, the cyanobacterium cell number decreases from (1.2 ± 0.2) × 106 to less than 104 cell mL-1, and the associated bacteria cell density decreases from (0.35 ± 0.03) × 106 to (1.3 ± 0.04) × 104 cell mL-1. Results indicate that the associated bacteria may not be removed even though 10.0 mg mL-1 of lysozyme is added into the non-axenic culture.
Table 2 Effects of lysozyme on the growth of cyanobacterium-associated bacteria and cyanobacterium
Lysozyme
(mg mL-1)
|
M. aeruginosa FACHB-905
(cell mL-1)
|
Cyanobacterium-associated bacteria
(cell mL-1)
|
0
1.0
|
2.2 × 106 ± 0.2 × 106
1.2 × 106 ± 0.2 × 106
|
0.64 × 106 ± 0.07 × 106
0.35 × 106 ± 0.03 × 106
|
2.0
|
0.75 × 106 ± 0.04 × 106
|
0.13 × 106 ± 0.01 × 106
|
5.0
10.0
|
Na
Na
|
3.2 × 104 ± 0.16 × 104
1.3 × 104 ± 0.04 × 104
|
|
|
|
|
Na represents not detected.
Isolation and purification of the axenic culture
The colony forming process of cyanobacterium and associated bacteria on solid plates (BG11 agar medium) is observed by inverted phase contrast microscope, and the results are shown in Fig. 3. It is obviously that the associated bacteria colonies are much bigger than the cyanobacterial colonies, indicating the associated bacteria are grew well than cyanobacterium. The cyanobacterium colony is formed when cultured for 15 d, although it is small; moreover, the cyanobacterial colonies are only found in 3 plates among the 20 replicate plates even after incubating for 20 d. The isolated cyanobacterial colonies are transferred into 6 test tubes and incubated for 3 d. The result shows that 5 tubes become green, indicating the cyanobacterium is grown well. However, the associated bacterium could still be detected. After two cycles of purification, only cyanobacterial colonies are observable on the BG11 solid medium. Possible contamination such as associated bacteria is subsequently examined before and after the incubations, and the results reveal that there is no contamination. Then a molecular identification is carried out for the purified axenic cyanobacterium named as M. aeruginosa FACHB-905A. The results indicate that M. aeruginosa FACHB-905A presented the highest sequence similarity (99% of identity) with M. aeruginosa NIES-843, M. aeruginosa PCC 7820 and M. aeruginosa PCC 7806.
Fig. 3 The growth of cyanobacterial and associated bacterial colonies (× 100). (a, b and c was the colonial morphology cultured for 1, 8 and 15 d, respectively)
Identification of associated bacteria
Two gram-negative bacteria, named B905-1 and B905-2, are isolated from the non-axenic M. aeruginosa FACHB-905. To identify the bacteria, phylogenetic analyses are performed using the 16S rDNA sequences. A total of 1367 bp of each of the two isolated strains is determined, and the 16S rDNA gene sequences obtained are subjected to GenBank BLAST search analyses. Strain B905-1 is most closely related to Pannonibacter phragmitetus L-s-R2A-19.4 with a 99% similarity value, and strain B905-2 is most closely related to Chryseobacterium sp. with a 97% similarity value. With the same method, another associated bacterium B907-1 is also successfully isolated from M. aeruginosa FACHB-907, and it is identified as Agrobacterium sp., which is most closely related to Agrobacterium sp. PNS-1 and Agrobacterium albertimagni C0008 with a 98% similarity value. The sequences of B905-1 and B907-1 are imported into the DNAMAN software V6 and aligned. Phylogenetic tree is then constructed (Fig. 4) and it is further confirmed that strain B905-1 and B907-1 are closely related to Pannonibacter sp. and Agrobacterium sp., respectively.
Fig. 4 The phylogenetic tree of cyanobacterium-associated bacteria.
Effect of associated bacteria on M. aeruginosa FACHB-905A
The growth rates of non-axenic culture (M. aeruginosa FACHB-905A) and axenic culture (M. aeruginosa FACHB-905A) are measured during both the static cultivation (without the shaking speed) and the shaking cultivation conditions (with the shaking speed of 150 rpm). Fig. 5 indicates that the generation time of axenic culture is 42.3 h (shaking cultivation) and 60.9 h (static cultivation), while the generation time of non-axenic culture is 33.6 h under the shaking cultivation and 45.3 h under the static cultivation, respectively. In addition, the generation time of non-axenic culture is much shorter than that of the axenic culture under the same cultivation condition, which demonstrates the photosynthetic efficiency of M. aeruginosa FACHB-905 is much better. At the same time, the growth rates of both the non-axenic culture and axenic culture under the shaking cultivation condition are much faster than that under the static cultivation condition. These results point to the role of the associated bacteria in promoting the growth of M. aeruginosa FACHB-905A.
Fig.5 The growth curves of axenic M. aeruginosa FACHB-905 and xenic M. aeruginosa FACHB-905A.
Effect of bacterium-cyanobacterium ratio on M. aeruginosa FACHB-905A
To further study the effect of associated bacterium B905-1 on the growth of axenic M. aeruginosa FACHB-905A, a series of experiments that different initial cyanobacterial cell concentrations with the bacterium-cyanobacterium ratio of 1:10 and 1:100 are undertaken in BG11 liquid medium (Fig. 6). Compared with the control group, the cyanobacterial cell numbers of treatment groups show a remarkable increase, while there is no obvious difference for the initial cyanobacterial cell number from 3.0 × 102 to 3.0 × 105 cell mL-1. It is (5.63 ± 0.08) × 106, (6.07 ± 0.15) × 106, (8.11 ± 0.25) × 106 and (11.75 ± 0.25) × 106 cell mL-1 for the treatment group of 1:10, while it is (4.47 ± 0.11) × 106, (3.98 ± 0.11) × 106, (4.92 ± 0.18) × 106 and (8.65 ± 0.44) × 106 cell mL-1 for that of 1:100 after incubating for 18 d, respectively. In addition, the higher cyanobacterial cell numbers of (14.72 ± 0.48) × 106 cell mL-1 for the treatment group of 1:10 and (10.63 ± 0.37) × 106 cell mL-1 for the treatment group of 1:100 are obtained at the day 21, respectively (Fig. 6d). These results indicate the addition of associated bacterium B905-1 has a positive promoting effect on the growth of M. aeruginosa FACHB-905A; besides, the bacterium-cyanobacterium ratio of 1:10 is much more suitable for promoting the growth of M. aeruginosa FACHB-905A than that of 1:100.
Fig. 6 Effects of bacterium-cyanobacterium ratio on axenic M. aeruginosa FACHB-905A (a, b, c and d show the initial algal cell number of 3.0 × 102, 3.0 × 103, 3.0 × 104 and 3.0 × 105 cell mL-1, respectively). * and ** represent a statistically significant difference of p < 0.05 and p < 0.01 when compared to the control.
The growth of axenic M. aeruginosa FACHB-905A on BG11 agar medium with and without the addition of strain B905-1 is also investigated. For the treatments that with the addition of strain B905-1, the cyanobacterial colony of axenic M. aeruginosa FACHB-905A becomes green after incubating for 20 days; while for the treatments that without the addition of strain B905-1, there is no cyanobacterial colony on BG11 agar medium. Moreover, the effects of different bacterium-cyanobacterium ratio (1:1, 1:10 and 1:100) on cyanobacterium division are studied (Fig. 7). Interestingly, the M. aeruginosa FACHB-905A is unable to grow in the treatment of 1:1 (Fig. 7b), but it grows well in both treatments of 1:10 and 1:100 (Fig. 7c and 7d). The results indicate high ratio of bacterium-cyanobacterium (1:1) is adverse to the growth of M. aeruginosa FACHB-905A.
Fig. 7 Effects of strain B905-1 on axenic M. aeruginosa FACHB-905A cultured by plate. (a was the control without strain B905-1; b, c and d show the treatments with the addition of strain B905-1 at an initial cell number of 1.0 × 104, 1.0 × 103 and 1.0 × 102 cell mL-1, respectively)
In order to prove that the promoting effect was associated with the extracellular substances of strain B905-1, the effect of the cell-free filtrate of strain B905-1 on the growth of M. aeruginosa FACHB-905A is carried out (Fig. 8). Results show that the cyanobacterium cell number of the treatment with the addition of the cell-free filtrate is 9.23 ± 0.56, 11.31 ± 1.85 and 22.14 ± 1.06 cell mL-1 after incubating for 4 d, 8 d and 12 d, respectively, and it is obviously higher than that with no cell-free filtrate. The axenic cyanobacterium grows much better with the addition of the cell-free filtrate again demonstrating strain B905-1 has the promoting effect on the growth of M. aeruginosa FACHB-905A; moreover, the released substances of strain B905-1 with the promoting effect apparently exist in the cell-free filtrate.
Fig. 8 Effect of cell-free filtrate of strain B905-1 on axenic M. aeruginosa FACHB-905A. * and ** represent a statistically significant difference of p < 0.05 and p < 0.01 as compared with the control.