In this ~ 300 m deep blue hole, dissolved oxygen (DO) decreased from 6.34 mg/L at 0 m to zero at 100 m, indicating that the environment above 100 m was oxic and below was anoxic; the concentration of DO dropped sharply (from 4.28 to 0.05 mg/L) between 80 and 100 m, indicating a DO-transition/suboxic zone. In addition, most physical and chemical parameters remained relatively uniform below the depth of 160 m (Yao et al. 2018). The water column could thus be divided into three zones; an oxic-suboxic zone (including the oxic zone and the suboxic zone, i.e., 0-90 m), a middle anoxic zone (100-160 m) and a bottom anoxic zone (160-230 m).
Water temperature decreased from 27.05 ℃ at the surface to 15.27 ℃, salinity increased from 33.4 to 34.6 PSU, while pH ranged from 7.26 to 8.14 (Table S1) (Yao et al. 2018). Generally, pH, DO and NO2- decreased with depth, whereas salinity, NH4+, PO43- and SiO32- showed opposite trends (Yao et al. 2018). DO was considered to be the main cause for these changes, while the other parameters, such as NH4+, NO3- and PO43-, co-varied with DO (Yao et al. 2018). Therefore, there are signifcant connections between DO and other environmental factors.
Because Vibrio spp. exhibit two alternative growth strategies, microorganisms were serially collected by 3-μm-pore size and 0.2-μm-pore size polycarbonate membranes, which were designated _F (free-living Vibrio) and _P (particleassociated Vibrio), respectively in the following analyses (Fig. 1).
Figure 1. Vertical section of SYBH at an azimuth of 30° (modifed from Xie et al. 2019)
The Vibrio population size ranged from 3.78 ×104 to 7.35 ×106 16S rRNA gene copies · L-1 in the blue hole seawater (Table 1). The highest abundance was measured at 100_F (7.35 ×106 gene copies · L-1) while the lowest was recorded at 170_P (3.78 ×104 gene copies · L-1). The abundance of free-living Vibrio population ranged from 1.21 × 105 to 7.35 ×106 gene copies · L-1, while the particle-associated Vibrio population varied from 3.78 ×104 to 9.08 × 105 gene copies · L-1. The abundance of free-living Vibrio population (~ 1.20 ×106 gene copies · L-1) was signifcantly higher than that of particle-associated Vibrio population (~ 2.68 × 105 gene copies · L-1) (P < 0.05) (Fig. 2). It was found that the abundance of free-living Vibrio population in the samples had a positive correlation to salinity, PO43- and SiO32-, and a negative correlation to DO and pH. No parameters showed signifcant correlation with the abundance of particle-associated Vibrio population (Table 2).
Free-living Vibrio Particle-associated Vibrio gene copies · L−1 Observed OTUs Chao 1 Shannon Coverage (%) gene copies · L−1 Observed OTUs Chao 1 Shannon Coverage (%) 0_F 6.76×105 40 66 1.342 99.96 0_P 7.84×105 36 38 1.264 99.98 10_F 2.54×105 69 69 2.089 100.00 10_P 7.11×104 60 60.75 1.608 99.99 40_F 3.72×105 39 39 1.514 100.00 40_P 4.68×104 33 33 1.381 100.00 70_F 1.21×105 29 29.5 1.419 99.99 70_P 1.51×105 23 23 1.356 100.00 80_F 1.69×105 34 35 1.492 100.00 80_P 7.72×104 20 21 0.978 99.99 85_F 7.89×105 25 25.5 1.428 99.99 85_P 1.69×105 25 25 1.618 100.00 90_F 8.75×105 32 32 1.613 100.00 90_P 9.47×104 26 26 1.420 100.00 100_F 7.35×106 18 18.3 0.474 99.99 100_P 8.68×105 26 26 1.402 100.00 120_F 9.30×105 27 30 1.321 99.99 120_P 9.75×104 24 24 1.709 100.00 140_F 1.45×106 37 40 1.444 99.99 140_P 9.08×105 32 32 1.741 100.00 170_F 1.79×106 54 60 1.799 99.99 170_P 3.78×104 33 33 0.943 100.00 190_F 4.21×105 65 66.5 2.199 99.99 190_P 1.01×105 41 41 1.284 100.00 230_F 3.77×105 66 66 2.018 100.00 230_P 8.30×104 45 46 1.313 99.99
Table 1. Richness and diversity estimates of total free-living and particle-associated Vibrio communities at SYBH based on 97% OTU clusters
Figure 2. The abundance of free-living and particle-associated Vibrio populations at diferent water depths of SYBH
Environmental factors Free-living Vibrio Particle-associated Vibrio Abundance Observed OTUs Chao 1 Shannon Abundance Observed OTUs Chao 1 Shannon Depth 0.473 0.099 0.072 0.291 0.027 0.124 0.124 -0.022 Temperature -0.473 -0.099 -0.072 -0.291 -0.027 -0.124 -0.124 0.022 Salinity 0.615* -0.045 -0.028 0.109 0.168 -0.006 -0.006 0.173 pH -0.559* -0.198 -0.193 -0.391 0.066 -0.247 -0.247 -0.008 DO -0.577* -0.057 -0.068 -0.226 -0.051 -0.091 -0.091 -0.147 NO3- -0.066 -0.554* -0.646* -0.264 -0.121 -0.605* -0.605* -0.129 NO2- -0.301 -0.657* -0.689** -0.546 0.218 -0.607* -0.607* 0.199 NH4+ 0.429 0.448 0.477 0.256 -0.036 0.503 0.503 -0.234 PO43- 0.571* 0.055 0.041 0.231 0.027 0.069 0.069 -0.104 SiO32- 0.571* 0.055 0.041 0.231 0.027 0.069 0.069 -0.104 Correlation coefcient is a positive value, indicating a positive correlation; correlation coefcient is a negative value, indicating a negative correlation
**Extremely signifcant related
Table 2. Correlation coefcients between abundance/diversity indices of Vibrio population and environmental factors at SYBH
The abundance of both the free-living and particle-associated Vibrio populations had no signifcant diferences among the three depth-zones (Kruskal-Wallis test, P > 0.05). Comparison of the relative proportions of Vibrio (i.e., proportion of Vibrio to total bacteria) in diferent sized fractions showed that Vibrio occur naturally and are an abundant group of marine microorganisms (0.15%-15.94% of total bacteria) in SYBH (Fig. S1).
The samples studied here yielded high-quality sequences ranging from 25, 645 to 43, 959 reads, of which 25, 094 sequences were left for each sample after normalizing. The average length of the obtained sequences was 512 base pairs. In total, 136 OTUs were clustered after randomly resampling, ranging from 18 to 69 OTUs per samples (Table 1), at a 97% similarity level. Good's coverages ranged from 99.96% to 100% in all the samples, indicating that most of the species in the study areas could be represented by the libraries generated by high-throughput sequencing.
Both the observed OTUs and Chao 1 index of free-living Vibrio were signifcantly higher than those of particle-associated Vibrio (P < 0.05, Wilcoxon's rank test), but there were no signifcant diferences in the Shannon index between them. Moreover, the observed OTUs were highest in the oxic-suboxic zone (102 OTUs), while the middle anoxic zone and bottom anoxic zone contained 49 and 97 OTUs, respectively. The Chao 1 index was signifcantly higher in the bottom anoxic zone than in the middle anoxic zone (P < 0.05). In addition, there were no signifcant diferences in diversity measures (including observed OTUs, Chao 1 index and Shannon index) between the oxic-suboxic zone and the middle anoxic zone (P > 0.05). The correlation coeffcients between diversity indices and environmental factors are shown in Table 2; the observed OTUs and Chao 1 index of free-living and particle-associated Vibrio communities had consistently negative relationships with NO3- and NO2-.
The taxonomy of each OTU was assigned against the Ezbiocloud database, with the 30 most abundant OTUs being shown in Fig. 3. Among the Vibrio OTUs (representing > 0.05% of the total normalized sequences), almost all sequences (98.89%) belonged to the Vibrionaceae family and 96.34% were assigned to the genus Vibrio. Seven OTUs (composed of all OTUs with > 1.0% of relative abundance in the total sequences) representing the most abundant species in the population, comprised 93.01% of the sequences among all samples. These seven OTUs included OTU47 (most similar to V. pelagius; accounting for 45.93%), OTU2 (most similar to V. harveyi; 22.40%), OTU55 (most similar to V. hepatarius 15.49%), OTU124 (most similar to V. sagamiensis; 3.91%), OTU134 (most similar to V. galatheae; 1.96%), OTU60 (1.88%; most similar to V. zhuhaiensis) and OTU119 (most similar to V. maritimus; 1.45%).
Figure 3. Vibrio community compositions at OTU level across all samples. _F and _P stand for free-living Vibrio and particle-associated Vibrio, respectively: (a) the most abundant 30 OTUs; (b) the most abundant 30 OTUs except the dominant 3 OTUs
The dominant groups difered signifcantly between each zone (Fig. 3); V. pelagius OTU47, V. harveyi OTU2, V. hepatarius OTU55 and V. sagamiensis OTU124 dominated the oxic-suboxic zone, V. hepatarius OTU55 and V. harveyi OTU2 dominated the middle anoxic zone, whereas V. pelagius OTU47 and V. harveyi OTU2 were the dominant groups in the bottom anoxic zone. To investigate the diferences in free-living and particle-associated Vibrio, the absolute abundance of dominant OTUs was calculated by multiplication of the total abundance of Vibrio and their relative proportions in the Vibrio communities (Table S3). It was found that the abundance of the dominant groups in the free-living fraction was greater than in the particle-associated fraction. No signifcant diferences were observed among the three depth zones for V. pelagius OTU47 and V. harveyi OTU2, but the abundance of V. hepatarius OTU55 was higher in the middle anoxic zone than in the other two depth zones.
While there were no marked diferences in the Vibrio community compositions between free-living and particleassociated fractions, signifcant vertical shifts were observed among the three depth-zones (Fig. 4a). The frst two principal components explained 49.89% of the total community variation. Clear variations between the depths below 170 m and other depths were found along the frst axis, whereas 0-90 m and 100-140 m could be separated by the second axis.
Figure 4. Community analysis of Vibrio and the relationship with environmental factors: (a) UniFrac principal coordinate analysis (PCoA) of diferent samples based on OTUs assigned at 97% sequence similarity; (b) db-RDA diagram illustrating the relationship between Vibrio community and environmental variables based on OTUs assigned at 97% sequence similarity. Blue, oxic-suboxic zone (0-90 m); red, middle anoxic zone (100-140 m); black, bottom anoxic zone (170-230 m). Dot, free-living Vibrio; square, particleassociated Vibrio; rhombus, free-living Vibrio and particle-associated had the same distribution in the db-RDA
The relationship between Vibrio community compositions and environmental factors at the OTU level was analyzed by db-RDA (Fig. 4b); RDA1 and RDA2 together explained 43.88% of the total variance among the samples. Two size fractions (_F and _P) at each depth were similar (Fig. 4b), so environmental factors shaped the same distribution of free-living and particle-associated Vibrio community at the same depth. Monte Carlo permutation tests showed that NH4+ (F = 9.3, P = 0.002), PO43- (F = 8.8, P = 0.002), SiO32- (F=7.2, P=0.002), temperature (F=6.4, P=0.002), pH (F = 6.4, P = 0.002), DO (F = 3.9, P = 0.002), salinity (F = 4.3, P = 0.004), and NO2- (F = 2.2, P = 0.03) signifcantly contributed to the Vibrio community structure.
Spearman's correlations between the relative abundance of the 30 most abundant OTUs and environmental factors were calculated. For both the free-living (Table S4) and particle-associated (Table S5) Vibrio communities, the three most dominant OTUs (V. pelagius OTU47, V. harveyi OTU2, and V. hepatarius OTU55) had no correlations with the tested environmental factors; many OTUs, which included most OTUs comprising < 4.0% of total sequences, demonstrated correlations with some of the environmental factors. For free-living Vibrio communities, the relative abundance of V. sagamiensis OTU124 was positively related to DO and NO3-, and negatively related to NH4+; V. zhuhaiensis OTU60 and V. maritimus OTU119 were negatively correlated with NO2- and DO, respectively. For particleassociated Vibrio communities, V. sagamiensis OTU124 was negatively correlated to depth, PO43-, SiO32- and salinity (P < 0.01), and positive correlated to temperature, DO, pH, and NO2- (P < 0.01). In addition, the relative abundance of V. zhuhaiensis OTU60 and V. maritimus OTU119 were negatively correlated with NO3- and NO2-, and NH4+ and DO, respectively. Within the top 30 OTUs, Vibrio sp. OTU61 and Vibrio sp. OTU45 occurred only in the anoxic water below 140 m, and Vibrio sp. OTU83 was present solely below 170 m.
To understand the ability of Vibrio spp. to degrade various macromolecules, an attempt was made to isolate Vibrio cultures from seawater at diferent depths of SYBH. From the two cruises, 19 species of Vibrionaceae (in total 178 isolates) were cultured, including V. antiquaries (79 isolates), V. neocaledonicus (19 isolates), V. parahaemolyticus (17 isolates), V. owensii (8 isolates), V. azureus (7 isolates), V. harveyi (6 isolates), V. tubiashii (5 isolates), V. alginolyticus (5 isolates), V. nigripulchritudo (4 isolates), V. japonicas (4 isolates), V. galatheae (2 isolates), V. maritimus (2 isolates), V. fuvialis (2 isolates), V. proteolyticus (1 isolate), V. hyugaensis (1 isolate), V. atypicus (1 isolate), Catenococcus thiocycli (10 isolates), Photobacterium damselae (1 isolate) and a potential new Vibrio species (4 isolates, isolated from the anaerobic microorganisms incubation media). All Vibrionaceae isolates (except the four potential new Vibrio isolates) and the depths isolated are shown in Fig. S2; the 16S rRNA genes of 91 isolates were sequenced and their GenBank accession numbers are shown in Table S6.
Media supplemented with a variety of macromolecules were used to detect extracellular enzymes. Of the 178 Vibrionaceae isolates cultured, 46 isolates consisting of 15 Vibrio species, were selected to test their extracellular enzymes, and 45 isolates were found to display at least one hydrolytic enzymatic activity. The ratio of hydrolysis circle diameter and colony diameter in the media indicated activity strength (Table S7). A total of 15 types of extracellular enzymatic activities detected at 28 ℃ were summarized in Fig. 5. Amylase, gelatinase, Tween 40 lipase and κ-carrageenanase producing strains (45 out of 46 isolates) were prevalent. Caseinase and lecithinase positive strains comprised a considerable proportion (39 and 38 out of 46 isolates, respectively), while none of the isolates displayed cellulolytic activity. The numbers of isolates that could degrade DNA, Tween 20 and Tween 80 at 28 ℃ were 35, 33 and 18, respectively. The numbers of isolates that were able to degrade chitin and alginate were 29 and 19, respectively. A V. owensii isolate cultured from the seawater at the depth of 170 m had no tested extracellular enzymes activities. Generally, Vibrio responded rapidly to all these macromolecules. For organic carbon sources readily available (including starch, Tween 20/40/80, gelatin, casein, DNA, lecithin, κ-carrageenan and hyaluronic acid), the transparent zones or hydrolytic circles could be observed after 1 day of culture. However, typically 1-2 weeks were required for other bacteria to utilize chitin (Zhang et al. 2016), while the transparent zones could be observed after 3 days' incubation.