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Subsurface karst caves provide unique opportunities to study the deep biosphere, shedding light on microbial contribution to elemental cycling. Although ammonia oxidation driven by both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) is well explored in soil and marine environments, our understanding in the subsurface biosphere still remained limited to date. To address this gap, weathered rock and sediment samples were collected from the Xincuntun Cave in Guilin City, an alkaline karst cave, and subjected to high-throughput sequencing and quantification of bacterial and archaeal amoA, along with determination of the potential nitrification rates (PNR). Results revealed that AOA dominated in ammonia oxidation, contributing 48–100% to the PNR, and AOA amoA gene copies outnumbered AOB by 2 to 6 orders. Nitrososphaera dominated in AOA communities, while Nitrosopira dominated AOB communities. AOA demonstrated significantly larger niche breadth than AOB. The development of AOA communities was influenced by deterministic processes (50.71%), while AOB communities were predominantly influenced by stochastic processes. TOC, NH4+, and Cl− played crucial roles in shaping the compositions of ammonia oxidizers at the OTU level. Cross-domain co-occurrence networks highlighted the dominance of AOA nodes in the networks and positive associations between AOA and AOB, especially in the inner zone, suggesting collaborative effort to thrive in extreme environments. Their high gene copies, dominance in the interaction with ammonia oxidizing bacteria, expansive niche breadth and substantial contribution to PNR collectively confirmed that AOA better adapted to alkaline, oligotrophic karst caves environments, and thus play a fundamental role in nitrogen cycling in subsurface biosphere.

Karst caves are usually considered as natural laboratories to study pristine microbiomes in subsurface biosphere. However, effects of the increasingly detected nitrate in underground karst ecosystem due to the acid rain impact on microbiota and their functions in subsurface karst caves have remained largely unknown. In this study, samples of weathered rocks and sediments were collected from the Chang Cave, Hubei province and subjected to high-throughput sequencing of 16S rRNA genes. The results showed that nitrate significantly impacted bacterial compositions, interactions, and functions in different habitats. Bacterial communities clustered according to their habitats with distinguished indicator groups identified for each individual habitat. Nitrate shaped the overall bacterial communities across two habitats with a contribution of 27.2%, whereas the pH and TOC, respectively, structured bacterial communities in weathered rocks and sediments. Alpha and beta diversities of bacterial communities increased with nitrate concentration in both habitats, with nitrate directly affecting alpha diversity in sediments, but indirectly on weathered rocks by lowering pH. Nitrate impacted more on bacterial communities in weathered rocks at the genus level than in sediments because more genera significantly correlated with nitrate concentration in weathered rocks. Diverse keystone taxa involved in nitrogen cycling were identified in the co-occurrence networks such as nitrate reducers, ammonium-oxidizers, and N2-fixers. Tax4Fun2 analysis further confirmed the dominance of genes involved in nitrogen cycling. Genes of methane metabolism and carbon fixation were also dominant. The dominance of dissimilatory and assimilatory nitrate reduction in nitrogen cycling substantiated nitrate impact on bacterial functions. Our results for the first time revealed the impact of nitrate on subsurface karst ecosystem in terms of bacterial compositions, interactions, and functions, providing an important reference for further deciphering the disturbance of human activities on the subsurface biosphere.

Karst ecosystems are widely distributed around the world, accounting for 15–20% of the global land area. However, knowledge on microbial ecology of these systems does not match with their global importance. To close this knowledge gap, we sampled three niches including weathered rock, sediment, and drip water inside the Heshang Cave and three types of soils overlying the cave (forest soil, farmland soil, and pristine karst soil). All these samples were subjected to high-throughput sequencing of V4-V5 region of 16S rRNA gene and analyzed with multivariate statistical analysis. Overall, archaeal communities were dominated by Thaumarchaeota, whereas Actinobacteria dominated bacterial communities. Thermoplasmata, Nitrosopumilaceae, Aenigmarchaeales, Crossiella, Acidothermus, and Solirubrobacter were the important predictor groups inside the Heshang Cave, which were correlated to NH4+ availability. In contrast, Candidatus Nitrososphaera, Candidatus Nitrocosmicus, Thaumarchaeota Group 1.1c, and Pseudonocardiaceae were the predictors outside the cave, whose distribution was correlated with pH, Ca2+, and NO2−. Tighter network structures were found in archaeal communities than those of bacteria, whereas the topological properties of bacterial networks were more similar to those of total prokaryotic networks. Both chemolithoautotrophic archaea (Candidatus Methanoperedens and Nitrosopumilaceae) and bacteria (subgroup 7 of Acidobacteria and Rokubacteriales) were the dominant keystone taxa within the co-occurrence networks, potentially playing fundamental roles in obtaining energy under oligotrophic conditions and thus maintaining the stability of the cave ecosystem. To be noted, all the keystone taxa of karst ecosystems were related to nitrogen cycling, which needs further investigation, particularly the role of archaea. The predicted ecological functions in karst soils mainly related to carbohydrate metabolism, biotin metabolism, and synthesis of fatty acid. Our results offer new insights into archaeal ecology, their potential functions, and archaeal interactions with bacteria, which enhance our understanding about the microbial dark matter in the subsurface karst ecosystems.

White spot lesions (WSLs) are common enamel infectious diseases in fixed orthodontic treatment, which might attribute to the dysbiosis of oral microbiome. However, the correlation of Candida albicans with oral bacteriome in WSLs still remains unrevealed. This study investigated the carriage of C. albicans and how it shaped the bacterial community in disease or healthy supragingival plaque, to explore the potential role of interkingdom interaction in orthodontic WSLs. In this study, 31 patients with WSLs (WSLs) and 23 healthy patients (Health) undergoing fixed orthodontic treatment were enrolled. The supragingival microbiota in both groups were determined using 16S rRNA gene sequencing. Colonization and abundance of C. albicans in the plaque were determined via culture-dependent and -independent methods. Among WSLs patients, the correlation of C. albicans and bacteriome was analyzed under QIIME2-based bioinformatics and Spearman’s correlation coefficient. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP404186). Significant differences in microbial diversity as well as composition were observed between WSLs and Health groups. Leptotrichia remarkably enriched in the WSLs group, while Neisseria and Cardiobacterium significantly enriched in the Health group. In addition, 45% of WSLs patients were C. albicans carriers but none in patients without WSLs. Among all WSLs patients, beta diversity and microbial composition were distinguished between C. albicans carriers and non-carriers. In C. albicans carriers, Corynebacterium matruchotii and Streptococcus mutans significantly enriched whereas Saccharibacteria_TM7_G-1 significantly depleted. The abundance of C. albicans was positively associated with bacteria such as Streptococcus mutans, while the negative correlation was detected between C. albicans and several bacteria such as Cardiobacterium hominis and Streptococcus sanguinis. Our study elucidated the distinguished supragingival plaque microbiome between orthodontic patients with and without WSLs. C. albicans frequently existed and enriched in orthodontic derived WSLs. The carriage of C. albicans shape plaque bacterial community in demineralized lesions and might play roles in WSLs pathogenesis.

Objective: To investigate the associations of Fc receptor-like 5 (FCRL5) gene polymorphisms and mRNA expression with liver fibrosis in chronic hepatitis B (CHB).Methods: A total of 114 CHB patients with liver fibrosis and 120 CHB patients without liver fibrosis were selected for this study. The gender, age, body mass index (BMI), alanine transaminase (ALT) value, aspartate aminotransferase (AST) value, aspartate aminotransferase-to-platelet ratio index (APRI), and fibrosis index based on 4 factors (FIB-4) were recorded. Two polymorphisms of the FCRL5 gene (rs6427384 and rs6692977) were genotyped. The mRNA expression level of FCRL5 in peripheral blood monocytes was determined.Results: ALT, AST, APRI, and FIB-4 in patients with fibrosis were significantly higher than those in non-fibrosis patients. There was statistically significant difference between fibrosis and non-fibrosis groups in the genotype distribution (χ2 = 7.805, p = 0.020) and allele frequencies (χ2 = 13.252, p < 0.001) at FCRL5 rs6692977. When compared with CC genotype, the genotype CT or TT at rs6692977 was significantly associated with a increased risk of liver fibrosis in CHB patients (CT vs. CC: OR = 1.921, 95% CI = 1.093–3.375, p = 0.023; TT vs. CC: OR = 2.598, 95% CI = 1.067–6.324, p = 0.031). The mRNA relative expression levels of FCRL5 in patients with liver fibrosis were significantly higher than those in the non-fibrosis group (t = 13.456, p

Karst caves are a natural oligotrophic subsurface biosphere widely distributed in southern China. Despite the progress in bacterial and fungal diversity, the knowledge about interactions between bacteria, fungi, and minerals is still limited in caves. Hence, for the first time, we investigated the interaction between bacteria and fungi living on weathered rocks in the Heshang Cave via high-throughput sequencing of 16S rRNA and ITS1 genes, and co-occurrence analysis. The mineral compositions of weathered rocks were analyzed by X-ray diffraction. Bacterial communities were dominated by Actinobacteria (33.68%), followed by Alphaproteobacteria (8.78%), and Planctomycetia (8.73%). In contrast, fungal communities were dominated by Sordariomycetes (21.08%) and Dothideomycetes (14.06%). Mineral substrata, particularly phosphorus-bearing minerals, significantly impacted bacterial (hydroxyapatite) and fungal (fluorapatite) communities as indicated by the redundancy analysis. In comparison with fungi, the development of bacterial communities was more controlled by the environmental selection indicated by the overwhelming contribution of deterministic processes. Co-occurrence network analysis showed that all nodes were positively linked, indicating ubiquitous cooperation within bacterial groups and fungal groups, as well as between bacteria and fungi under oligotrophic conditions in the subsurface biosphere. In total, 19 bacterial ASVs and 34 fungal OTUs were identified as keystone taxa, suggesting the fundamental role of fungi in maintaining the microbial ecosystem on weathered rocks. Ascomycota was most dominant in keystone taxa, accounting for 26.42%, followed by Actinobacteria in bacteria (24.53%). Collectively, our results confirmed the highly diverse bacterial and fungal communities on weathered rocks, and their close cooperation to sustain the subsurface ecosystem. Phosphorus-bearing minerals were of significance in shaping epipetreous bacterial and fungal communities. These observations provide new knowledge about microbial interactions between bacteria, fungi, and minerals in the subterranean biosphere.

The level of water table and temperature are two environmental variables shaping soil bacterial communities, particularly in peatland ecosystems. However, discerning the specific impact of these two factors on bacterial communities in natural ecosystems is challenging. To address this issue, we collected pore water samples across different months (August and November in 2017 and May 2018) with a gradient of water table changes and temperatures at the Dajiuhu peatland, Central China. The samples were analyzed with 16S rRNA high-throughput sequencing and Biolog EcoMicroplates. Bacterial communities varied in the relative abundances of dominant taxa and harbored exclusive indicator operational taxonomic units across the different months. Despite these differences, bacterial communities showed high similarities in carbon utilization, with preferences for esters (pyruvic acid methyl ester, Tween 40, Tween 80, and D-galactonic acid γ-lactone), amino acids (L-arginine and L-threonine), and amines (phenylethylamine and putrescine). However, rates of carbon utilization (as indicated by average well-color development) and metabolic diversity (McIntosh and Shannon index) in May and August were higher than those in November. Redundancy analysis revealed that the seasonal variations in bacterial communities were significantly impacted by the level of the water table, whereas the temperature had a fundamental role in bacterial carbon utilization rate. Co-occurrence analysis identified Sphingomonas, Mucilaginibacter, Novosphingobium, Lacunisphaera, Herminiimonas, and Bradyrhizobium as keystone species, which may involve in the utilization of organic compounds such as amino acids, phenols, and others. Our findings suggest that bacterial community functions were more stable than their compositions in the context of water table changes. These findings significantly expand our current understanding of the variations of bacterial community structures and metabolic functions in peatland ecosystems in the context of global warming and fluctuation of the water table.

Sphagnum-associated microbiomes are crucial to Sphagnum growth and peatland ecological functions. However, roles of rare species in bacterial communities across Sphagnum compartments are poorly understood. Here the structures of rare taxa (RT) and conditionally abundant and rare taxa (CART) from Sphagnum palustre peat (SP), S. palustre ectosphere (Ecto) and S. palustre endosphere (Endo) were investigated in the Dajiuhu Peatland, central China. Our results showed that plant compartment effects significantly altered the diversities and structures of bacterial communities. The Observed species and Simpson indices of RT and CART in alpha diversity significantly increased from Endo to SP, with those of Ecto in-between. The variations of community dissimilarities of RT and CART among compartments were consistent with those of whole bacterial communities (WBC). Network analysis indicated a non-random co-occurrence pattern of WBC and all keystone species are affiliated with RT and CART, indicating their important role in sustaining the WBC. Furthermore, the community structures of RT and CART in SP were significantly shaped by water table and total nitrogen content, which coincided with the correlations between WBC and environmental factors. Collectively, our results for the first time confirm the importance of rare species to bacterial communities through structural and predicted functional analyses, which expands our understanding of rare species in Sphagnum-associated microbial communities in subalpine peatlands.

Sphagnum microbiomes play an important role in the northern peatland ecosystems. However, information about above and belowground microbiomes related to Sphagnum at subtropical area remains largely limited. In this study, microbial communities from Sphagnum palustre peat, S. palustre green part, and S. palustre brown part at the Dajiuhu Peatland, in central China were investigated via 16S rRNA gene amplicon sequencing. Results indicated that Alphaproteobacteria was the dominant class in all samples, and the classes Acidobacteria and Gammaproteobacteria were abundant in S. palustre peat and S. palustre brown part samples, respectively. In contrast, the class Cyanobacteria dominated in S. palustre green part samples. Microhabitat differentiation mainly contributes to structural differences of bacterial microbiome. In the S. palustre peat, microbial communities were significantly shaped by water table and total nitrogen content. Our study is a systematical investigation on above and belowground bacterial microbiome in a subalpine Sphagnum peatland and the results offer new knowledge about the distribution of bacterial microbiome associated with different microhabitats in subtropical area.

In the past several decades, sulfate concentration and salinity have been considered to be the two essential hydrochemical factors in the formation of dolomite, yet arguments against this hypothesis have existed simultaneously. To clarify the effects of sulfate concentration and salinity in the mineralization of dolomite, we conducted experiments on dolomite precipitation mediated by a halophilic archaeon, Natrinema sp. J7-1 with various sulfate concentrations and salinities. This strain was cultured in a series of modified growth media (MGM) with salinities of 140, 200, and 280‰. Cells in the post-log phase were harvested and used to mediate the formation of dolomite in solutions with various sulfate concentrations of 0, 3, 29.8, and 100 mM and salinities of 140, 200, and 280‰. X-ray diffraction (XRD) spectra showed that proto-dolomite, monohydrocalcite, and aragonite formed in samples with cells, yet only aragonite was detected in samples without cells. Proto-dolomite was found in all biotic samples, regardless of the variation in salinity and sulfate concentration. Moreover, the relative abundances of proto-dolomite in the precipitates were positively correlated with the salinities of the media but were uncorrelated with the sulfate concentrations of the solutions. Scanning electronic microscopy (SEM) and energy dispersive spectroscopy (EDS) results showed that all the proto-dolomites were sphere or sphere aggregates with a mole ratio of Mg/Ca close to 1.0. No obvious variations in morphology and Mg/Ca were found among samples with various sulfate concentrations or salinities. This work reveals that a variation of sulfate concentration in solution (from 0 to 100 mM) does not affect the formation of dolomite mediated by halophilic archaea, but an increase of salinity (from 140 to 280‰) enhances this process. Our results indicate that under natural conditions, an increase in salinity may be more significant than the decrease of sulfates in microbe-mediated dolomite formation.

Deciphering of the mycobiome in pristine karst caves has been impeded by constraints of remote locations, inaccessibility to specimens and technical limitations, which greatly restricted in-depth understanding of mycobiomes in subterranean ecosystem. Here, mycobiomes of Heshang Cave in south-western karst region of China were investigated by Illumina HiSeq sequencing of fungal rRNA-ITS1 gene across different habitats. In total 793,502 ITS1 reads and 2,179 OTUs from 778 Mb reads after stringent quality control (Q30) and 453 genera, 72 orders and 19 classes within 6 phyla were detected. Ascomycota (42% OTUs) dominated across the five habitats. Shannon-Wiener index varied from 1.25 to 7.62 and community richness was highest in drip waters, followed by weathered rocks, bat guanos, sediments, and air samples. Mycobiomes displayed specificity to five habitats and more distinct OTUs were found in weathered rocks (12%) and drip waters (9%). In contrast, only 6.60% core OTUs were shared by five habitats. Notably, weathered rocks possessed more indicator groups and were revealed for the first time to be dominated by Sordariomycetes (43%). The community richness of air mycobiomes increased from cave entrance to the innermost part and dominated by the indicator groups of Penicillium mallochii (>30%) and P. herquei (>9%). Our work represents the largest attempt to date to a systematical investigation of oligotrophic solution-cave-associated mycobiomes in China. Our discovery of high diversity of mycobiomes in Heshang Cave also suggests that eukaryotic microorganisms may play a crucial role in subsurface environments.

Enhanced monsoon duration and soil acidification from acid rain, are expected to impact the distribution of microbial communities of surface and subsurface environments, although these impacts are poorly understood for most systems. In central China, soluble carbonate bedrock forms extensive karst landscapes. Current predictions are that the amount of monsoonal precipitation and acid rainfall in central China will increase, which is expected to lead to changes in the pH balance of karst ecosystems. To evaluate the role of pH, TOC and other geochemical parameters (e.g., Ca2+, Mg2+, NH4+, NOx, SO42-) in shaping bacterial communities within a single karst system in central China, samples were collected from the thin surface soils overlying Heshang cave, cave sediments, and weathered cave passage rocks from the entrance, twilight, and dark zones, as well as from epikarstic drip waters inside the cave. Illumina sequencing of 16S rRNA genes and multivariate statistical analyses revealed that each tested community was distinct and the community variability was significantly correlated with pH, total organic carbon, and potassium concentrations. Specifically, surface soils were dominated by Acidobacteria, Verrucomicrobia and Planctomycetes, and diversity significantly decreased with acidic pH values. Nitrospirae, Gemmatimonadetes, Firmicutes, and Chloroflexi were unique to cave sediments, while Actinobacteria and Proteobacteria dominated weathered rocks and drip waters, respectively. The results reveal important implications regarding the effects of acidification on bacterial communities in karst areas, and on the control of pH in shaping bacterial communities throughout a karst system. Increased water flux into and through karst habitats due to monsoonal precipitation may result in deeper penetration of acidic solutions into karst and shift the bacterial communities inside the cave in the future.

Caves are nutrient-limited and dark subterranean ecosystems. To date, attention has been focused on geological research of caves in China, whilst indigenous microbial diversity has been insufficiently characterized. Here, we report the fungal diversity in the pristine, oligotrophic, karst Heshang Cave, central China, using a culture-dependent method coupled with the analysis of the fungal rRNA-ITS gene sequences. A total of 194 isolates were obtained with six different media from 14 sampling sites of sediments, weathered rocks, and bat guanos. Phylogenetic analysis clustered the 194 sequenced isolates into 33 genera within 15 orders of three phyla, Ascomycota, Basidiomycota, and Zygomycota, indicating a high degree of fungal diversity in the Heshang Cave. Notably, 16 out of the 36 fungal genera were also frequently observed in solution caves around the world and 23 genera were previously found in carbonate cave, indicating potential similarities among fungal communities in cave ecosystems. However, 10 genera in this study were not reported previously in any solution caves, thus expanding our knowledge about fungal diversity in cave ecosystems. Moreover, culturable fungal diversity varied from one habitat to another within the cave, being the highest in sediments, followed by weathered rocks and bat guanos as indicated by a-diversity indexes. At the genus level, Penicillium accounted for 40, 54, and 52% in three habitats of sediments, weathered rocks, and bat guanos, respectively. Trichoderma, Paecilomyces, and Aspergillus accounted for 9, 22, and 37% in the above habitats, correspondingly. Despite of the dominance of Penicillium in all samples, β-diversity index indicated significant differences between each two fungal communities in the three habitats in view of both the composition and abundance. Our study is the first report on fungal communities in a natural pristine solution cave system in central China and sheds light on fungal diversity and functions in cave ecosystems. [ABSTRACT FROM AUTHOR]