A Comprehensive Census of Microbial Diversity in Hot Springs of Tengchong, Yunnan Province China Using 16S rRNA Gene Pyrosequencing

Abstract

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21–123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5–2.6), high temperature (85.1–89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6–4.8) and cooler temperature (55.1–64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2–9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii”, and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.

Humboldt’s spa: microbial diversity is controlled by temperature in geothermal environments

“…Singletons remained in the analysis, and these studies are therefore compared with our unfiltered data containing singletons. Hou et al (2013) denoised their data set, whereas Cole et al (2013) did not, however our analyses suggest that denoising has a minimal effect on the Shannon index (on average a 15.7% decline; Supplementary Table S2). Alpha diversities for these two studies were rarefied to depths of 978 and 5325, both of which are above the 700-sequence depth at which our samples leveled off.…”

“…To further verify the temperature-diversity relationship, Shannon diversity index data from two recent pyrotag sequencing studies of neutralalkaline hot springs in China and the US (Cole et al, 2013;Hou et al, 2013) were included in Figure 3b. To conform with our sample set, only sediment and biomat samples were included, whereas water samples were ignored.…”

“…Primers used by Cole et al (2013) are the same primers used in the current study and amplified the V6-V8 region of the 16S rRNA gene. Primers from Hou et al (2013) amplified the V4-V8 region. Both the studies used Mothur to remove low quality reads and downstream analyses were performed using QIIME.…”

“…The lack of consensus is not surprising given the lack of statistical robustness and the possibility for site-specific effects in these studies. They may also have suffered from a focus on samples with temperatures 455 1C (Everroad et al, 2012;Cole et al, 2013;Hou et al, 2013) where the relationship is weakest. Decreasing alpha diversity with increasing temperature was most evident in our sample set from 25-65 1C, but above this the relationship flattened out compared with experimental noise (Figure 3, Supplementary Figure S1).…”

“…However, studies comparing diversity across geothermal systems remain few in number and consistent trends remain unresolved. Although some studies have noted a lack of correlation (Yim et al, 2006;Huang et al, 2011;Hou et al, 2013;Wang et al, 2013) or even a slight increase in diversity with increasing temperature (Purcell et al, 2007), others have noted a decrease in diversity with increasing temperature (Miller et al, 2009;Tobler and Benning, 2011;Everroad et al, 2012;Cole et al, 2013). Although these studies hint at underlying general patterns, they are all limited by at least one of several shortcomings: restricted methodologies that detect only the few most abundant operational taxonomic units (OTUs), such as construction of clone libraries (Yim et al, 2006;Purcell et al, 2007;Miller et al, 2009;Huang et al, 2011;Tobler and Benning, 2011;Everroad et al, 2012); low numbers of samples (o10) (Yim et al, 2006;Purcell et al, 2007;Miller et al, 2009;Tobler and Benning, 2011;Everroad et al, 2012); samples taken from a single geothermal field and therefore possibly showing site-specific trends (Yim et al, 2006;Miller et al, 2009;Everroad et al, 2012;Cole et al, 2013); or a limited temperature range (o30 1C total range) (Purcell et al, 2007;Miller et al, 2009;Everroad et al, 2012;Cole et al, 2013).…”