Liuqin Huang

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

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.

Control of Temperature on Microbial Community Structure in Hot Springs of the Tibetan Plateau

The Tibetan Plateau in Northwest China hosts a number of hot springs that represent a biodiversity hotspot for thermophiles, yet their diversity and relationship to environmental conditions are poorly explored in these habitats. In this study we investigated microbial diversity and community composition in 13 Tibetan hot springs with a wide range of temperatures (22.1–75°C) and other geochemical conditions by using the 16S rRNA gene pyrosequencing approach. Bacteria (108–1011 copy/g; 42 bacterial phyla) in Tibetan hot springs were more abundant and far more diverse than Archaea (107–1010 copy/g; 5 archaeal phyla). The dominant bacterial phyla systematically varied with temperature. Moderate temperatures (75–66°C) favored Aquificae, GAL35, and novel Bacteria, whereas low temperatures (60–22.1°C) selected for Deinococcus-Thermus, Cyanobacteria, and Chloroflexi. The relative abundance of Aquificae was correlated positively with temperature, but the abundances of Deinococcus-Thermus, Cyanobacteria, and Chloroflexi were negatively correlated with temperature. Cyanobacteria and Chloroflexi were abundant in Tibetan hot springs and their abundances were positively correlated at low temperatures (55–43°C) but negatively correlated at moderate temperatures (75–55°C). These correlation patterns suggest a complex physiological relationship between these two phyla. Most archaeal sequences were related to Crenarchaeota with only a few related to Euryarchaeota and Thaumarchaeota. Despite the fact that microbial composition in Tibetan hot springs was strongly shaped by temperature, microbial diversity (richness, evenness and Shannon diversity) was not significantly correlated with temperature change. The results of this study expand our current understanding of microbial ecology in Tibetan hot springs and provide a basis for a global comparison.

Production of branched tetraether lipids in the lower Pearl River and estuary: effects of extraction methods and impact on bGDGT proxies

Branched glycerol dibiphytanyl glycerol tetraethers (bGDGTs) are known as bacterial lipids that occur widely in terrestrial environments, particularly in anaerobic peat bogs and soil. We examined the abundance and distribution of bGDGTs in both core (C) and polar (P) lipid fractions from the water column and surface sediments in the lower Pearl River (PR) and its estuary using two extraction methods (sonication vs. Bligh and Dyer). A number of soil samples in the lower PR drainage basin were also collected and extracted for bGDGTs using the sonication method. The results showed aquatic production of bGDGTs as supported by substantial abundances of P-bGDGTs in the water column and sediment samples. The bGDGT-based proxies (BIT, CBT, and MBT) were not affected by the method of extraction when C-bGDGTs were analyzed; in such case, the pHCBT of the sediments reflected the soil pH of the lower PR drainage basin, and the temperature close to the annual mean air temperature (MAT) in the lower PR basin. On the other hand, the P-bGDGT-derived proxies were inconsistent between the two methods. The P-bGDGTs (particularly those extracted using the sonication method) may not be reliable indicators of annual MATs.

Latitudinal Distribution of Ammonia-Oxidizing Bacteria and Archaea in the Agricultural Soils of Eastern China

ABSTRACT The response of soil ammonia-oxidizing bacterial (AOB) and archaeal (AOA) communities to individual environmental variables (e.g., pH, temperature, and carbon- and nitrogen-related soil nutrients) has been extensively studied, but how these environmental conditions collectively shape AOB and AOA distributions in unmanaged agricultural soils across a large latitudinal gradient remains poorly known. In this study, the AOB and AOA community structure and diversity in 26 agricultural soils collected from eastern China were investigated by using quantitative PCR and bar-coded 454 pyrosequencing of the amoA gene that encodes the alpha subunit of ammonia monooxygenase. The sampling locations span over a 17° latitude gradient and cover a range of climatic conditions. The Nitrosospira and Nitrososphaera were the dominant clusters of AOB and AOA, respectively; but the subcluster-level composition of Nitrosospira-related AOB and Nitrososphaera-related AOA varied across the latitudinal gradient. Variance partitioning analysis showed that geography and climatic conditions (e.g., mean annual temperature and precipitation), as well as carbon-/nitrogen-related soil nutrients, contributed more to the AOB and AOA community variations (∼50% in total) than soil pH (∼10% in total). These results are important in furthering our understanding of environmental conditions influencing AOB and AOA community structure across a range of environmental gradients.

Greater temporal changes of sediment microbial community than its waterborne counterpart in Tengchong hot springs, Yunnan Province, China

Temporal variation in geochemistry can cause changes in microbial community structure and diversity. Here we studied temporal changes of microbial communities in Tengchong hot springs of Yunnan Province, China in response to geochemical variations by using microbial and geochemical data collected in January, June and August of 2011. Greater temporal variations were observed in individual taxa than at the whole community structure level. Water and sediment communities exhibited different temporal variation patterns. Water communities were largely stable across three sampling times and dominated by similar microbial lineages: Hydrogenobaculum in moderate-temperature acidic springs, Sulfolobus in high-temperature acidic springs, and Hydrogenobacter in high-temperature circumneutral to alkaline springs. Sediment communities were more diverse and responsive to changing physicochemical conditions. Most of the sediment communities in January and June were similar to those in waters. However, the August sediment community was more diverse and contained more anaerobic heterotrophs than the January and June: Desulfurella and Acidicaldus in moderate-temperature acidic springs, Ignisphaera and Desulfurococcus in high-temperature acidic springs, the candidate division OP1 and Fervidobacterium in alkaline springs, and Thermus and GAL35 in neutral springs. Temporal variations in physicochemical parameters including temperature, pH, and dissolved organic carbon may have triggered the observed microbial community shifts.

Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria in Diverse Chinese Paddy Soils

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) in three types of paddy soils of China before and after rice plantation were investigated by using an integrated approach including geochemistry, 454 pyrosequencing, and quantitative polymerase chain reaction (PCR). The abundances of AOA amoA gene were 1∼2 orders of magnitude higher than AOB amoA gene. The types of paddy soils had important impacts on the diversities of both AOA and AOB via clay mineralogy (smectite or illite-rich) and bioavailability of ammonium. The Nitrososphaera subcluster 5 and Nitrosopumilis cluster of AOA, and Nitrosomonas subcluster 5 and Nitrosospira subcluster 3 of AOB were well adapted to soils with high ammonium concentrations. AOA and AOB community structures were different before and after rice plantation, likely due to changes of pH and ammonium fertilization. The Nitrosospira subclusters 2 and 9 were well adapted to acidic paddy soils. However, the sensitivity of AOA and AOB community structures to these factors may be complicated by other geochemical conditions. The results of this study collectively demonstrated that multiple environmental factors, such as clay mineralogy, ammonium content and total organic carbon as well as soil pH, shaped AOA and AOB community structure and abundance.

Cultivation and characterization of thermophilic Nitrospira species from geothermal springs in the US Great Basin, China, and Armenia

Despite its importance in the nitrogen cycle, little is known about nitrite oxidation at high temperatures. To bridge this gap, enrichment cultures were inoculated with sediment slurries from a variety of geothermal springs. While nitrite-oxidizing bacteria (NOB) were successfully enriched from seven hot springs located in US Great Basin, south-western China, and Armenia at ≤ 57.9 °C, all attempts to enrich NOB from > 10 hot springs at ≥ 61 °C failed. The stoichiometric conversion of nitrite to nitrate, chlorate sensitivity, and sensitivity to autoclaving all confirmed biological nitrite oxidation. Regardless of origin, all successful enrichments contained organisms with high 16S rRNA gene sequence identity (≥ 97%) with Nitrospira calida. In addition, Armenian enrichments also contained close relatives of Nitrospira moscoviensis. Physiological properties of all enrichments were similar, with a temperature optimum of 45-50 °C, yielding nitrite oxidation rates of 7.53 ± 1.20 to 23.0 ± 2.73 fmoles cell(-1) h(-1), and an upper temperature limit between 60 and 65 °C. The highest rates of NOB activity occurred with initial NO2 - concentrations of 0.5-0.75 mM; however, lower initial nitrite concentrations resulted in shorter lag times. The results presented here suggest a possible upper temperature limit of 60-65 °C for Nitrospira and demonstrate the wide geographic range of Nitrospira species in geothermal environments.

Distribution and diversity of aerobic carbon monoxide- oxidizing bacteria in geothermal springs of China, the Philippines, and the United States

Accumulating genomic evidence suggests that a variety of thermophilic bacteria contain cox operons and may be capable of aerobic carbon monoxide (CO) oxidation. However, little is known about the distribution and diversity of the cox-encoding (COXE) bacteria in natural geothermal environments. In this study, we examined coxL gene (encoding the large subunit of carbon monoxide dehydrogenase: CoxL) sequences retrieved from the sediments of 25 geothermal sites located in the Qinghai-Tibetan Plateau (QTP) and Yunnan Province (YP) of China, the Bacon-Manito Geothermal Production Field (BGPF) of the Philippines, and the Great Basin of the United States (USGB). Temperature and pH ranges of the studied hot springs were 22.1 to 90.8°C and 2.7 to 9.4, respectively. Phylogenetic analyses showed that most CoxL sequences were closely related to the classes Actinobacteria, Deinococci, Ktedonobacteria, Thermomicrobia, and Clostridia, and hot springs from different regions hosted different COXE communities. In addition, these hot springs harbored some COXE bacteria that were phylogenetically distinct from those inhabiting nongeothermal ecosystems. This study revealed no significant correlation between temperature or pH and the composition or diversity of COXE communities at the global scale. However, within a given region, temperature was correlated with the COXE bacterial community composition.

Actinobacterial Diversity in the Sediments of Five Cold Springs on the Qinghai-Tibet Plateau.

The actinobacterial diversity was investigated in the sediments of five cold springs in Wuli region on the Qinghai-Tibet Plateau using 16S rRNA gene phylogenetic analysis. The actinobacterial communities of the studied cold springs were diverse and the obtained actinobacterial operational taxonomic units were classified into 12 actinobacterial orders (e.g., Acidimicrobiales, Corynebacteriales, Gaiellales, Geodermatophilales, Jiangellales, Kineosporiales, Micromonosporales, Micrococcales, Nakamurellales, Propionibacteriales, Pseudonocardiales, Streptomycetales) and unclassified Actinobacteria. The actinobacterial composition varied among the investigated cold springs and were significantly correlated (r = 0.748, P = 0.021) to environmental variables. The actinobacterial communities in the cold springs were more diverse than other cold habitats on the Tibetan Plateau, and their compositions showed unique geographical distribution characteristics. Statistical analyses showed that biogeographical isolation and unique environmental conditions might be major factors influencing actinobacterial distribution among the investigated cold springs.

Thioarsenate Formation Coupled with Anaerobic Arsenite Oxidation by a Sulfate-Reducing Bacterium Isolated from a Hot Spring

Thioarsenates are common arsenic species in sulfidic geothermal waters, yet little is known about their biogeochemical traits. In the present study, a novel sulfate-reducing bacterial strain Desulfotomaculum TC-1 was isolated from a sulfidic hot spring in Tengchong geothermal area, Yunnan Province, China. The arxA gene, encoding anaerobic arsenite oxidase, was successfully amplified from the genome of strain TC-1, indicating it has a potential ability to oxidize arsenite under anaerobic condition. In anaerobic arsenite oxidation experiments inoculated with strain TC-1, a small amount of arsenate was detected in the beginning but became undetectable over longer time. Thioarsenates (AsO4-xSx2- with x = 1–4) formed with mono-, di- and tri-thioarsenates being dominant forms. Tetrathioarsenate was only detectable at the end of the experiment. These results suggest that thermophilic microbes might be involved in the formation of thioarsenates and provide a possible explanation for the widespread distribution of thioarsenates in terrestrial geothermal environments.