Cyanobacterial Community Structure and Isolates From Representative Hot Springs of Yunnan Province, China Using an Integrative Approach

Abstract

Cyanobacteria from the representative hot springs of Yunnan Province, China are explored for their diversity and community composition following an integrative approach of cultivation-independent and -dependent studies and further isolation of potential taxa for future biotechnological perspective. 16S rRNA amplicon sequencing of microbial mats in these hot springs with temperature ranging from 38 to 90°C revealed Cyanobacteria and Proteobacteria constituting a bounteous portion of the bacterial community. The combined approach of 16S rRNA amplicon sequencing and phenotypic analysis revealed the diversity of cyanobacteria (a total of 45 genera). Out of these, a total of 19 cyanobacterial taxa belonging to 6 genera and 10 species were isolated as individuals with the possibility of biotechnological utilization. These isolates were subjected to a thorough morphological study and molecular characterization using 16S rRNA gene sequencing for identification and understanding their phylogeny. The identity and phenotypic and genotypic characteristics of 7 cyanobacterial isolates are not identical to any known cyanobacterial species, generating scope for future taxonomic novelties. Preliminary experiments based on high-temperature (50°C) cultivation showed that most of the isolates were thermotolerant and suggested for their high biotechnological usage potential.

Keywords: 16S rRNA amplicon sequencing; cyanobacteria; hot springs; phenotype; phylogenetics.

FIGURE 1

Location of sampling sites (marked with red triangles) in Yunnan Province, China. Geographic coordinates were determined using a Garmin Etrex GPS. Digital Elevation Model (DEM) data information was downloaded from Resource and Environment data cloud Platform of China (http://www.resdc.cn/). Pictures in the right panel indicate the surrounding sampling environment of the cyanobacterial biofilms in different hot springs, 1: Dongshan-001, 2–3: Huangpo-002 (soil), 4–7: Huangpo-003 (4 samples), 8: Rehai (Hamazui)-004, 9: Rehai (Hamazui)-005, 10: Rehai (Yanjingquan)-006, 11: Rehai (Zhenzhuquan)-007, 12: Rehai (Dagunguo)-008. 13–14: Rehai (Dagunguo)-009, 15: Banglazhang-011, 16–17: Banglazhang-012, 18: Banglazhang-013, 19: Banglazhang-014, 20: Banglazhang-015, 21: Banglazhang-016.

FIGURE 2

OTU Venn diagram illustrating distribution of cyanobacterial OTUs among the four groups of temperature range (A) and three groups of pH range (B). T indicates the temperatures of the water samples or the hot spring water temperatures of other sample types. T30–50 (Group 1: 30–50°C), T50–70 (Group 2: 50–70°C), T70–80 (Group 3: 70–80°C), and T80–90 (Group 4: 80–90°C). The Venn diagram was generated using the OmicStudio tools (https://www.omicstudio.cn/tool).

FIGURE 3

The Shannon–Weaver and Simpson indices of cyanobacterial OTUs in thermal springs based on temperature range (A,B) and location (C,D). The Box plot was generated using the Origin tools. Median values and interquartile ranges have been indicated in the plots. T indicates the temperatures of the water samples or the hot spring water temperatures of other sample types. T30–50 (Group 1: 30–50°C), T50–70 (Group 2: 50–70°C), T70–80 (Group 3: 70–80°C), and T80–90 (Group 4: 80–90°C).

FIGURE 4

The cyanobacterial OTU abundance clustering heat map. The rows and columns represent the OTU IDs and the sampling sites (A) temperature groups (B), respectively. The OTU clustering tree is in the left. The value of each square color of the middle heat map corresponds to the relative abundance of each row of OTU. The heat map was generated using the OmicStudio tools (https://www.omicstudio.cn/tool).

FIGURE 5

Cyanobacterial genera found based on the 16S rRNA amplicon sequencing analysis (A) and microscopic observation (B) of the samples collected from different studied sites of hot springs/hot water source of China. A pie chart was included in each histogram to display the percentage of cyanobacterial strains based on morphological subsections.

FIGURE 6

Mat-forming cyanobacteria identified in this study. (A) Synechococcus cf. nidulans, (B) Gloeocapsa gelatinosa, (C) Chlorogloeopsis fritschii, (D) Anabaena sp. NK1-14, (E) Fischerella sp. NK 1-16, (F) Fischerella sp. NK 1-20, (G) Fischerella thermalis, (H) Planktothrix cryptovaginata, (I) Leptolyngbya copelandii, (J) L. dangeardii, (K) L. faveolarum, (L) Leptolyngbya sp. NK 1-10, (M) Leptolyngbya sp. NK 1-23, (N) Leptolyngbya sp. NK 1-12, (O) Thermoleptolyngbya oregonensis, (P) Leptolyngbya thermobia, (Q) L. valderiana.

FIGURE 7

Verification of the thermotolerant feature of the hot spring cyanobacterial isolates. Note that the isolated cyanobacterial species was grown statically in 10-ml test tubes at 50°C under 30 μmol photon m^–2 s^–1 light intensity for 10 days.

FIGURE 8

Phylogenetic relationships (Neighbor-Joining) between the isolated cyanobacteria species of hot springs of Yunnan Province, China and other closest relative hot spring cyanobacteria based on 16S rRNA gene sequences. Isolated cyanobacteria were indicated in red color. The number near the node represents the bootstrap value. Subsection was mentioned in brackets. Gloeobacter violaceus PCC 7421 (AF132790) was used as an out-group.