16S amplicon sequencing of microbial communities in enriched and non-enriched sediments of non-volcanic hot spring with temperature gradients

Abstract

Microorganisms in geothermal springs can offer insights into the fundamental and applied study of extremophiles. However, low microbial abundance and culturing requirements limit the ability to analyze microbial diversity in these ecosystems. In this study, culture-dependent and culture-independent techniques were used to analyze sediment samples from the non-volcanic Tatta Pani hot springs in district Poonch of Azad Kashmir. Microbial composition, temperature gradient, and enrichment effects on rare taxa were evaluated. In total, 31 distinct bacterial phyla and 725 genera were identified from the non-enriched Tatta Pani hot spring sediment samples, and 33 distinct bacterial phyla and 890 genera from the enriched sediment samples. Unique phyla specimens from the enriched samples included Candidatus Cloacimonetes, Caldiserica, and Korarchaeota archaea. The enriched samples yielded specific microbiota including 805 bacteria and 42 archaea operational taxonomic units with 97% similarity, though decreased thermophilic microbiota were observed in the enriched samples. Microbial diversity increased as temperature decreased. Candidate novel species were isolated from the culture-dependent screening, along with several genera that were not found in the 16S amplicon sequencing data. Overall, the enriched sediments showed high microbial diversity but with adverse changes in the composition of relatively dominant bacteria. Metagenomic analyses are needed to study the diversity, phylogeny, and functional investigation of hot spring microbiota.

Keywords: 16S amplicon sequencing; Culture-dependent; Enrichment; Hot spring; Tatta Pani; Thermophile.

Figure 1. Map and images of the Tatta Pani hot spring.

(A) The sample location is overlaid on the Sentinel-2 image obtained through free public access from Sentinel-2 multispectral satellite images provided by the European Space Agency (© European Space Agency). (B) Images of the sample collection sites at different temperatures.

Figure 2. Multivariate analysis of the samples from Tatta Pani hot spring.

(A) Principal coordinate analysis and (B) hierarchical clustering using Bray-Curtis dissimilarity analysis revealed a shift in the microbial community with temperature and enrichment changes. The alphabetical letters in the sample name represent the sample group, and the numeric value indicates the temperature in centigrade. TP, sediment samples from Tatta Pani hot spring; ETP, enriched sediment samples from Tatta Pani hot spring.

Figure 3. Hierarchical clustering of six relatively abundant phyla in the enriched and nonenriched samples from Tatta Pani hot spring, using the unweighted pair group method with arithmetic mean tree in the Bray–Curtis analysis.

The bar chart represents the relative abundance of phyla in percentages (A) Proteobacteria, (B) Firmicutes, (C) Nitrospirae, (D) Aquificae, (E) Acidobacteria, and (F) Chloroflexi. The alphabetical letters in the sample name represent the sample group, and the numeric value indicates the temperature in centigrade. TP, sediment samples from Tatta Pani hot spring; ETP, enriched sediment samples from Tatta Pani hot spring.

Figure 4. Comparative analysis of 20 most abundant families in TP and ETP sample of different temperature.

The bar chart represents the relative abundance of families in percentages between TP and ETP samples collected at temperature gradients (A) 64 °C, (B) 60 °C, (C) 58 °C, (D) 55 °C, (E) 45 °C, and (F) 20 °C. The alphabetical letters in sample name represent sample group and numeric value indicate temperature in centigrade. TP, sediment samples from Tatta Pani hot spring; ETP, enriched sediment samples from Tatta Pani hot spring.

Figure 5. Heatmap showing percentage relative abundance (≥1%) of genera in at least one enriched or non-enriched sample from Tatta Pani hot spring.

Sample clustering was performed based on the Pearson correlation. The alphabetical letters in the sample name represent the sample group, and the numeric value indicates the temperature in centigrade. TP, sediment samples from Tatta Pani hot spring; ETP, enriched sediment samples from Tatta Pani hot spring.

Figure 6. Analysis of common and unique genera identified from culture-dependent analysis of the Tatta Pani hot spring sample TP64 (TP64-C), compared with 16S amplicon sequence analysis of non-enriched TP64 sample (TP64-16S).

(A) The circular nodes in the network attached with two edges indicate common genera between TP64-C and TP64-16S. (B) The percentage of relative abundance of the common genera between TP64-C and TP64-16S.