3.1 Seaweeds Inhibition to the V. anguillarum 65 under different conditions
To the selected seaweed from 5 g to 25 g in the tests, its inhibition to V. anguillarum 65 was significantly hight. On the first 1-2 days, the number of V. anguillarum in the 25 g co-cultured groups was significantly decreased in the 5 g co-cultured systems (Fig. S1), it reflected that increase of seaweed (U. pertusa and G. lemaneiformis) could enhance the inhibitions of V. anguillarum 65.
Under continuous culture in dark, seaweeds exhibited less inhibitions to V. anguillarum 65, there was obvious inhibition effect when under both continuous illumination and 12:12 (L/D), and the inhibition effect under continuous illumination was significantly stronger than that in irradiances 12:12 (L/D) (Fig. S2), this implied that light is crucial to seaweed physiological activities for inhibiting to V. anguillarum 65, and increasing illumination could enhance the inhibition to V. anguillarum 65.
One interesting phenomena was that, after supplying of NaNO3 and KH2PO4 into the seawater without seaweeds, the number of V. anguillarum 65 was higher than that of tested group without nitrogen and phosphorus nutrients supplying, this indicated that NaNO3 and KH2PO4 could either enhance the growth of V. anguillarum 65 (Fig. S3). While the adding NaNO3 and KH2PO4 to the seawater contained with V. anguillarum 65 and seaweeds, the seaweeds inhibition effects on V. anguillarum 65 were significantly higher (Fig. S3), this implied that the NaNO3 and KH2PO4 could enhance the inhibitory effect of seaweed on V. anguillarum 65.
When removing the seaweeds from the co-culture system in 3 days, the inhibition effects to V. anguillarum 65 became less, the antibacterial effect of seawater with retained microorganisms is slightly better than that in sterilized seawater (Fig. S4). This indicated that the seaweeds were the main retarding factors to V. anguillarum 65, and the inhibition effect of microorganisms, metabolites and other factors in seawater to V. anguillarum 65 was limited, while the inhibition Vibrio was not enhanced by Vibrio itself (Fig. S5). The co-culture of V. anguillarum 65 and seaweeds before experiment was applied for stimulating the seaweeds to enhance its tests ability. Besides, the ability of seaweed to inhibit Vibrio was not been enhanced by Vibrio stimulation (Fig. S5). This indicated that seaweed inhibition of Vibrio is an inherent ability, the co-cultivation of Vibrio and seaweeds can not enhance seaweeds antibacterial ability.
3.2 The difference of inhibition effects of several seaweeds on Vibrio
The bacteriostatic tests for the 8 types of seaweeds on the 3 Vibrio strains were examined, and the bacteriostatic test results of 3 species of Vibrio exhibited highly similarities (Fig. 2; Fig. S6; S7). The detected chlorophyll fluorescence parameter Fv/Fm decreased with the time passed by, and were believed as the damage of continuous illumination due to nutrient depletion in the cultured system (Fig. 2a; Fig. S6a; S7a). The detected DO value exhibited raised first and then decreased (Fig. 2b; Fig. S6b; S7b). When culture for 9 hrs, the DO detected reached the maxim, and then declined until 48hrs. Besides, the detected pH value (Fig. 2c; Figs. S6c; S7c) increased continuously in 24 hrs, and then reached to stable. All the seaweeds showed the inhibitions on Vibrio (Fig. 2d, Fig. S6d; S7), and the inhibition of U. prolifera and U. pertusa was obvious higher than that of other seaweeds, and showed Vibrio reductions below 10 CFU/mL in 48 hrs.
To the 8 types of seaweeds for 24 hrs (Fig. S8), the detected DO, pH value and Vibrio number exhibited highly relations to each other. The higher DO and pH values were, the less Vibrio number was, we concluded that the increase of dissolved oxygen and pH value could indeed inhibit Vibrio.
3.3 Dissolved oxygen and pH on the Vibrio growth
The inhibition effects on Vibrio were testified, and it showed with U. pertusa > DO 25 mg/L > cultured G. lemaneiformis > DO 20 mg/L > DO 15 mg/L > DO 10 mg/L (Fig. 3a; Fig S9a; Fig S10a). When the dissolved oxygen content in the seawater reached to 10 mg/L, it would inhibit Vibrio growth, with the increase of oxygen, the inhibition to Vibrio was significantly enhanced. The inhibitory effect of U. pertusa (dissolved oxygen content reached to 24.46 mg/L) on Vibrio was more obvious than that of 25 mg/L, and the antibacterial effect of cultivated G. lemaneiformis (highest dissolved oxygen content reached to 21.15 mg/L) would less obvious than 20 mg/L. Effects of pH and two types of seaweeds on the inhibition to Vibrio were U. pertusa> cultured G. lemaneiformis> pH 9.6 > pH 9.2 > pH 8.8 > pH 8.4 (Fig. 3b; Fig S9b; Fig S10b). When the pH reached to 8.4, it showed less inhibitory effect on Vibrio, the increase of pH enhanced the inhibitory effect for Vibrio. When the pH reached to 9.6, the inhibitory effect for Vibrio was close to that of G. lemaneiformis (maximum pH was 9.35). When pH and dissolved oxygen were co-increased, the inhibitory effect on Vibrio was more significant. The inhibition effect of DO value (20 mg/L) and pH value (9.2) was similar to that of G. lemaneiformis (Fig. 3c, Fig S9c and Fig S10c). When the DO value reached to 25 mg/L, and pH value was at 9.6, the inhibition effect for Vibrio could inhibit the growth of U. pertusa.
3.4 Microbial diversity analysis
To the analysis of 21 seaweeds’ microbial biodiversities, totally there were 79,980 pairs of reads obtained, and it contained average 79,841 clean reads after quality control and splicing examining. The removal of chimeras yielded an average of 50,579 effective reads per sample (Table S1).
3.4.1 ASV analysis
ASVs (amplification sequence variants) represent the true biological sequences, and show the microbial species in the samples. The average ASV number of the samples in GB, UB, GA, UA, W, GW and UW were 3598, 3330, 2899, 2552, 1874, 366 and 387, respectively (Fig. S11). After 3 days culturing, the symbiotic and epiphytic microorganisms of ASV on the two seaweeds decreased by 19.4% and 23.6% respectively. Moreover, the ASV number of seawater microorganisms decreased 80.5% and 79.3% respectively after the seaweeds were co-cultured for 3 days.
3.4.2 Alpha diversity analysis
After 3 days of culturing with seaweeds, the ACE, Chao1, Simpson and Shannon indexes on seaweeds symbiotic and epiphytic microorganisms were lower than that of symbiotic and epiphytic microorganisms on seaweeds before the culturing (Table 3). These parameters decreased significantly after the culturing. After 3 days of culturing, the diversities of symbiotic and epiphytic microorganisms and the microorganisms in the seawater were significantly decreased, and caused some microorganisms disappeared, because these microorganisms were not able to adapt to the seawater environments under the high dissolved oxygen and high pH conditions. Before and after 3 days of co-culture, the detected ACE, Chao1, Simpson and Shannon indexes on G. lemaneiformis were higher than that of U. pertusa, it indicated that the symbiotic and epiphytic microorganisms of G. lemaneiformis showed higher diversities. The microbial diversity in the seawater of G. lemaneiformis decreased more significantly than that of U. pertusa.
Table 3 Alpha diversity index statistics of different samples
Sample ID
|
ACE
|
Chao1
|
Simpson
|
Shannon
|
Coverage
|
GB
|
3600.06
|
3597.76
|
1.00
|
10.59
|
1.00
|
UB
|
3334.12
|
3330.50
|
1.00
|
10.38
|
1.00
|
GA
|
2904.85
|
2899.34
|
1.00
|
10.10
|
1.00
|
UA
|
2556.26
|
2552.17
|
1.00
|
9.64
|
1.00
|
W
|
1876.33
|
1874.15
|
0.99
|
8.67
|
1.00
|
GW
|
369.66
|
367.31
|
0.90
|
5.49
|
1.00
|
UW
|
390.60
|
388.14
|
0.93
|
5.71
|
1.00
|
3.4.3 Analysis of significant differences between the microorganisms
The microbial classification with significant difference (p<0.05) were adopted for further analysis. From the phylum level, the symbiotic and epiphytic microbial community on seaweeds was not variable drastically before and after three days of cultivation, the relative epiphytic microbial abundances on the G. lemaneiformis decreased from the phylum, such as Firmicutes, Fusobacteria, Myxococcus, Cyanobacteria, etc. However, the relative abundance of Bacteroidota and Patescibacteria increased in 3 days of culture (Fig. S12a). The symbiotic and epiphytic microorganism of U. pertusa had little change at the phylum level. Only the relative abundance of Cyanobacteria decreased significantly after cultivation, and the hydrophytes disappeared, the abundance of Dependentiae increased (Fig. S12b). The relative abundance of some symbiotic and epiphytic microorganisms on the G. lemaneiformis on genus increased, it included unclassified Rhodobacteraceae, Vibrio, unclassified Micavibrionaceae, unclassified Candidatus, Kaiserbacillus, Epibacillus, etc. The decreased abundances groups are: Maribus, Fusobacterium, RB41, one unclassified Lachnospiraceae, etc (Fig. S13a). The relative abundance of Altermonadaceae, Altermonas, Marivita, Terasakiella and Algicola in the symbiotic and epiphytic microorganisms of U. pertusa increased; the relative abundance of Vibrio and Acinetobacter decreased (Fig. S13b).
The microorganisms in seawater with seaweeds changed dramatically before and after the co-culturing. From phylum considerations, the relative abundance of Proteobacteria (highest abundance) increased after culturing, the relative abundance of Campylobacter increased in seawater for culturing of U. ertusa, while the relative abundance of microorganisms decreased, and some microorganisms completely disappeared in the seawater after culturing, such as Elusimicrobiota, Latescibacillus, etc. (Fig. S14a). At the genus considerations, the microorganisms showed significant differences (p<0.05), the relative abundance of most genera in seawater microorganisms after cultivation of seaweeds also increased compared with that before cultivation of seaweeds, including unclassified Rhodobacter, Nautella, Polaribacter, Jannaschia and Marivita. The relative abundance of some genera declined or even disappeared completely, such as Clade_ Ia, Candidatus Actinomarina, NS5 marine group, etc. (Fig. S14b). It implied that the testified microorganism could tolerate high dissolved oxygen and high pH, but the relative abundance of microorganisms that could not tolerate high dissolved oxygen and high pH in the seawater.
3.4.4 FAPROTAX ecological function prediction
The FAPROTAX database was adopted for predicting the function of microbial communities. The chemical energy heterotrophic type was the mainly aerobic microorganism and increased in the co-culture system, and it was consist with the increase of dissolved oxygen in the co-culturing (Fig. 4).
3.4.5 Detection of Vibrio quantities and relative abundance
The relative abundance of Vibrio increased in the seawater when culturing of seaweeds, the relative abundance of symbiotic and epiphytic Vibrio in G. lemaneiformis increased after 3 days of culturing, the relative abundance of symbiotic and epiphytic Vibrio in U. pertusa decreased in 3 days (Table 4). The amount of Vibrio decreased significantly in the seawater and on the surface of seaweeds after 3 days of culturing. The increase in relative abundance and decrease in absolute quantity may be caused by a significant decrease in the total amount of microorganisms, this might be caused by the increase of dissolved oxygen content and pH value which caused by seaweeds. This mean that seaweeds could not only show inhibition effect on Vibrio, but also showed inhibition effect to other microorganisms which are not tolerant to high dissolved oxygen and high pH conditions.
Table 4 Relative abundance and absolute quantity of Vibrio in the 5 tested groups
Sample
|
W
|
GW
|
UW
|
GB
|
GA
|
UB
|
UA
|
Relative abundance of Vibrio (%)
|
0.13
|
2.25
|
0.41
|
0.73
|
2.41
|
1.00
|
0.57
|
The number of Vibrio (CFU/mL)
|
21500
|
15
|
<10
|
53600
|
335
|
43780
|
112
|