BIOCOM-PIPE: a new user-friendly metabarcoding pipeline for the characterization of microbial diversity from 16S, 18S and 23S rRNA gene amplicons.

BACKGROUND: The ability to compare samples or studies easily using metabarcoding so as to better interpret microbial ecology results is an upcoming challenge. A growing number of metabarcoding pipelines are available, each with its own benefits and limitations. However, very few have been developed to offer the opportunity to characterize various microbial communities (e.g., archaea, bacteria, fungi, photosynthetic microeukaryotes) with the same tool. RESULTS: BIOCOM-PIPE is a flexible and independent suite of tools for processing data from high-throughput sequencing technologies, Roche 454 and Illumina platforms, and focused on the diversity of archaeal, bacterial, fungal, and photosynthetic microeukaryote amplicons. Various original methods were implemented in BIOCOM-PIPE to (1) remove chimeras based on read abundance, (2) align sequences with structure-based alignments of RNA homologs using covariance models, and (3) a post-clustering tool (ReClustOR) to improve OTUs consistency based on a reference OTU database. The comparison with two other pipelines (FROGS and mothur) and Amplicon Sequence Variant definition highlighted that BIOCOM-PIPE was better at discriminating land use groups. CONCLUSIONS: The BIOCOM-PIPE pipeline makes it possible to analyze 16S, 18S and 23S rRNA genes in the same packaged tool. The new post-clustering approach defines a biological database from previously analyzed samples and performs post-clustering of reads with this reference database by using open-reference clustering. This makes it easier to compare projects from various sequencing runs, and increased the congruence among results. For all users, the pipeline was developed to allow for adding or modifying the components, the databases and the bioinformatics tools easily, giving high modularity for each analysis.

The diversity of soil microbial communities matters when legumes face drought.

The cultivation of legumes shows promise for the development of sustainable agriculture, but yield instability remains one of the main obstacles for its adoption. Here, we tested whether the yield stability (i.e., resistance and resilience) of pea plants subjected to drought could be enhanced by soil microbial diversity. We used a dilution approach to manipulate the microbial diversity, with a genotype approach to distinguish the effect of symbionts from that of microbial diversity as a whole. We investigated the physiology of plants in response to drought when grown on a soil containing high or low level of microbial diversity. Plants grown under high microbial diversity displayed higher productivity and greater resilience after drought. Yield losses were mitigated by 15% on average in the presence of high soil microbial diversity at sowing. Our study provides proof of concept that the soil microbial community as a whole plays a key role for yield stability after drought even in plant species living in relationships with microbial symbionts. These results emphasize the need to restore soil biodiversity for sustainable crop management and climate change adaptation.

High Microbial Diversity Promotes Soil Ecosystem Functioning.

In soil, the link between microbial diversity and carbon transformations is challenged by the concept of functional redundancy. Here, we hypothesized that functional redundancy may decrease with increasing carbon source recalcitrance and that coupling of diversity with C cycling may change accordingly. We manipulated microbial diversity to examine how diversity decrease affects the decomposition of easily degradable (i.e., allochthonous plant residues) versus recalcitrant (i.e., autochthonous organic matter) C sources. We found that a decrease in microbial diversity (i) affected the decomposition of both autochthonous and allochthonous carbon sources, thereby reducing global CO2 emission by up to 40%, and (ii) shaped the source of CO2 emission toward preferential decomposition of most degradable C sources. Our results also revealed that the significance of the diversity effect increases with nutrient availability. Altogether, these findings show that C cycling in soil may be more vulnerable to microbial diversity changes than expected from previous studies, particularly in ecosystems exposed to nutrient inputs. Thus, concern about the preservation of microbial diversity may be highly relevant in the current global-change context assumed to impact soil biodiversity and the pulse inputs of plant residues and rhizodeposits into the soil.IMPORTANCE With hundreds of thousands of taxa per gram of soil, microbial diversity dominates soil biodiversity. While numerous studies have established that microbial communities respond rapidly to environmental changes, the relationship between microbial diversity and soil functioning remains controversial. Using a well-controlled laboratory approach, we provide empirical evidence that microbial diversity may be of high significance for organic matter decomposition, a major process on which rely many of the ecosystem services provided by the soil ecosystem. These new findings should be taken into account in future studies aimed at understanding and predicting the functional consequences of changes in microbial diversity on soil ecosystem services and carbon storage in soil.

The interaction of soil phototrophs and fungi with pH and their impact on soil CO2, CO18O and OCS exchange.

The stable oxygen isotope composition of atmospheric CO2 and the mixing ratio of carbonyl sulphide (OCS) are potential tracers of biospheric CO2 fluxes at large scales. However, the use of these tracers hinges on our ability to understand and better predict the activity of the enzyme carbonic anhydrase (CA) in different soil microbial groups, including phototrophs. Because different classes of the CA family (α, ß and γ) may have different affinities to CO2 and OCS and their expression should also vary between different microbial groups, differences in the community structure could impact the 'community-integrated' CA activity differently for CO2 and OCS. Four soils of different pH were incubated in the dark or with a diurnal cycle for forty days to vary the abundance of native phototrophs. Fluxes of CO2, CO18O and OCS were measured to estimate CA activity alongside the abundance of bacteria, fungi and phototrophs. The abundance of soil phototrophs increased most at higher soil pH. In the light, the strength of the soil CO2 sink and the CA-driven CO2-H2O isotopic exchange rates correlated with phototrophs abundance. OCS uptake rates were attributed to fungi whose abundance was positively enhanced in alkaline soils but only in the presence of increased phototrophs. Our findings demonstrate that soil-atmosphere CO2, OCS and CO18O fluxes are strongly regulated by the microbial community structure in response to changes in soil pH and light availability and supports the idea that different members of the microbial community express different classes of CA, with different affinities to CO2 and OCS.

Soil C and N models that integrate microbial diversity.

Industrial agriculture is yearly responsible for the loss of 55-100 Pg of historical soil carbon and 9.9 Tg of reactive nitrogen worldwide. Therefore, management practices should be adapted to preserve ecological processes and reduce inputs and environmental impacts. In particular, the management of soil organic matter (SOM) is a key factor influencing C and N cycles. Soil microorganisms play a central role in SOM dynamics. For instance, microbial diversity may explain up to 77 % of carbon mineralisation activities. However, soil microbial diversity is actually rarely taken into account in models of C and N dynamics. Here, we review the influence of microbial diversity on C and N dynamics, and the integration of microbial diversity in soil C and N models. We found that a gain of microbial richness and evenness enhances soil C and N dynamics on the average, though the improvement of C and N dynamics depends on the composition of microbial community. We reviewed 50 models integrating soil microbial diversity. More than 90 % of models integrate microbial diversity with discrete compartments representing conceptual functional groups (64 %) or identified taxonomic groups interacting in a food web (28 %). Half of the models have not been tested against an empirical dataset while the other half mainly consider fixed parameters. This is due to the difficulty to link taxonomic and functional diversity.

Urban land uses shape soil microbial abundance and diversity.

Soil microbial biodiversity provides many useful services in cities. However, the ecology of microbial communities in urban soils remains poorly documented, and studies are required to better predict the impact of urban land use. We characterized microbial communities (archea/bacteria and fungi) in urban soils in Dijon (Burgundy, France). Three main land uses were considered - public leisure, traffic, and urban agriculture - sub-categorized in sub-land uses according to urban indexes and management practices. Microbial biomass and diversity were determined by quantifying and high-throughput sequencing of soil DNA. Variation partitioning analysis was used to rank soil physicochemical characteristics and land uses according to their relative contribution to the variation of soil microbial communities. Urban soils in Dijon harbored high levels of microbial biomass and diversity that varied according to land uses. Microbial biomass was 1.8 times higher in public leisure and traffic sites than in urban agriculture sites. Fungal richness increased by 25 % in urban agriculture soils, and bacterial richness was lower (by 20 %) in public leisure soils. Partitioning models explained 25.7 %, 46.2 % and 75.6 % of the variance of fungal richness, bacterial richness and microbial biomass, respectively. The organic carbon content and the C/N ratio were the best predictors of microbial biomass, whereas soil bacterial diversity was mainly explained by soil texture and land use. Neither metal trace elements nor polycyclic aromatic hydrocarbons contents explained variations of microbial communities, probably due to their very low concentration in the soils. The microbial composition results highlighted that leisure sites represented a stabilized habitat favoring specialized microbial groups and microbial plant symbionts, as opposed to urban agriculture sites that stimulated opportunistic populations able to face the impact of agricultural practices. Altogether, our results provide evidence that there is scope for urban planners to drive soil microbial diversity through sustainable urban land use and associated management practices.

Biogeographical patterns of the soil fungal:bacterial ratio across France.

Soils are one of the major reservoirs of biological diversity on our planet because they host a huge richness of microorganisms. The fungal:bacterial (F:B) ratio targets two major functional groups of organisms in soils and can improve our understanding of their importance and efficiency for soil functioning. To better decipher the variability of this ratio and rank the environmental parameters involved, we used the French Soil Quality Monitoring Network (RMQS)-one of the most extensive and a priori-free soil sampling surveys, based on a systematic 16 km × 16 km grid and including more than 2,100 samples. F:B ratios, measured by quantitative PCR targeting the 18S and 16S rDNA genes, turned out to be heterogenously distributed and spatially structured in geographical patterns across France. These distribution patterns differed from bacterial or fungal densities taken separately, supporting the hypothesis that the F:B ratio is not the mere addition of each density but rather results from the complex interactions of the two functional groups. The F:B ratios were mainly influenced by soil characteristics and land management. Among soil characteristics, the pH and, to a lesser extent, the organic carbon content and the carbon:nitrogen (C:N) ratio were the main drivers. These results improved our understanding of soil microbial communities, and from an operational point of view, they suggested that the F:B ratio should be a useful new bioindicator of soil status. The resulting dataset can be considered as a first step toward building up a robust repository essential to any bioindicator and aimed at guiding and helping decision making. IMPORTANCE In the face of human disturbances, microbial activity can be impacted and, e.g., can result in the release of large amounts of soil carbon into the atmosphere, with global impacts on temperature. Therefore, the development and the regular use of soil bioindicators are essential to (i) improve our knowledge of soil microbial communities and (ii) guide and help decision makers define suitable soil management strategies. Bacterial and fungal communities are key players in soil organic matter turnover, but with distinct physiological and ecological characteristics. The fungal:bacterial ratio targets these two major functional groups by investigating their presence and their equilibrium. The aim of our study is to characterize this ratio at a territorial scale and rank the environmental parameters involved so as to further develop a robust repository essential to the interpretation of any bioindicator of soil quality.

Inferring microbiota functions from taxonomic genes: a review.

Deciphering microbiota functions is crucial to predict ecosystem sustainability in response to global change. High-throughput sequencing at the individual or community level has revolutionized our understanding of microbial ecology, leading to the big data era and improving our ability to link microbial diversity with microbial functions. Recent advances in bioinformatics have been key for developing functional prediction tools based on DNA metabarcoding data and using taxonomic gene information. This cheaper approach in every aspect serves as an alternative to shotgun sequencing. Although these tools are increasingly used by ecologists, an objective evaluation of their modularity, portability, and robustness is lacking. Here, we reviewed 100 scientific papers on functional inference and ecological trait assignment to rank the advantages, specificities, and drawbacks of these tools, using a scientific benchmarking. To date, inference tools have been mainly devoted to bacterial functions, and ecological trait assignment tools, to fungal functions. A major limitation is the lack of reference genomes-compared with the human microbiota-especially for complex ecosystems such as soils. Finally, we explore applied research prospects. These tools are promising and already provide relevant information on ecosystem functioning, but standardized indicators and corresponding repositories are still lacking that would enable them to be used for operational diagnosis.

Potential of Meta-Omics to Provide Modern Microbial Indicators for Monitoring Soil Quality and Securing Food Production.

Soils are fundamental resources for agricultural production and play an essential role in food security. They represent the keystone of the food value chain because they harbor a large fraction of biodiversity-the backbone of the regulation of ecosystem services and "soil health" maintenance. In the face of the numerous causes of soil degradation such as unsustainable soil management practices, pollution, waste disposal, or the increasing number of extreme weather events, it has become clear that (i) preserving the soil biodiversity is key to food security, and (ii) biodiversity-based solutions for environmental monitoring have to be developed. Within the soil biodiversity reservoir, microbial diversity including Archaea, Bacteria, Fungi and protists is essential for ecosystem functioning and resilience. Microbial communities are also sensitive to various environmental drivers and to management practices; as a result, they are ideal candidates for monitoring soil quality assessment. The emergence of meta-omics approaches based on recent advances in high-throughput sequencing and bioinformatics has remarkably improved our ability to characterize microbial diversity and its potential functions. This revolution has substantially filled the knowledge gap about soil microbial diversity regulation and ecology, but also provided new and robust indicators of agricultural soil quality. We reviewed how meta-omics approaches replaced traditional methods and allowed developing modern microbial indicators of the soil biological quality. Each meta-omics approach is described in its general principles, methodologies, specificities, strengths and drawbacks, and illustrated with concrete applications for soil monitoring. The development of metabarcoding approaches in the last 20 years has led to a collection of microbial indicators that are now operational and available for the farming sector. Our review shows that despite the recent huge advances, some meta-omics approaches (e.g., metatranscriptomics or meta-proteomics) still need developments to be operational for environmental bio-monitoring. As regards prospects, we outline the importance of building up repositories of soil quality indicators. These are essential for objective and robust diagnosis, to help actors and stakeholders improve soil management, with a view to or to contribute to combining the food and environmental quality of next-generation farming systems in the context of the agroecological transition.

Correction: Mapping and predictive variations of soil bacterial richness across France.

[This corrects the article DOI: 10.1371/journal.pone.0186766.].

In situ dynamics and spatial heterogeneity of soil bacterial communities under different crop residue management.

The effect of the location of wheat residues (soil surface vs. incorporated in soil) on their decomposition and on soil bacterial communities was investigated by the means of a field experiment. Bacterial-automated ribosomal intergenic spacer analysis of DNA extracts from residues, detritusphere (soil adjacent to residues), and bulk soil evidenced that residues constitute the zone of maximal changes in bacterial composition. However, the location of the residues influenced greatly their decomposition and the dynamics of the colonizing bacterial communities. Sequencing of 16S rRNA gene in DNA extracts from the residues at the early, middle, and late stages of degradation confirmed the difference of composition of the bacterial community according to the location. Bacteria belonging to the γ-subgroup of proteobacteria were stimulated when residues were incorporated whereas the α-subgroup was stimulated when residues were left at the soil surface. Moreover, Actinobacteria were more represented when residues were left at the soil surface. According to the ecological attributes of the populations identified, our results suggested that climatic fluctuations at the soil surface select populations harboring enhanced catabolic and/or survival capacities whereas residues characteristics likely constitute the main determinant of the composition of the bacterial community colonizing incorporated residues.

In situ dynamics of microbial communities during decomposition of wheat, rape, and alfalfa residues.

Microbial communities are of major importance in the decomposition of soil organic matter. However, the identities and dynamics of the populations involved are still poorly documented. We investigated, in an 11-month field experiment, how the initial biochemical quality of crop residues could lead to specific decomposition patterns, linking biochemical changes undergone by the crop residues to the respiration, biomass, and genetic structure of the soil microbial communities. Wheat, alfalfa, and rape residues were incorporated into the 0-15 cm layer of the soil of field plots by tilling. Biochemical changes in the residues occurring during degradation were assessed by near-infrared spectroscopy. Qualitative modifications in the genetic structure of the bacterial communities were determined by bacterial-automated ribosomal intergenic spacer analysis. Bacterial diversity in the three crop residues at early and late stages of decomposition process was further analyzed from a molecular inventory of the 16S rDNA. The decomposition of plant residues in croplands was shown to involve specific biochemical characteristics and microbial community dynamics which were clearly related to the quality of the organic inputs. Decay stage and seasonal shifts occurred by replacement of copiotrophic bacterial groups such as proteobacteria successful on younger residues with those successful on more extensively decayed material such as Actinobacteria. However, relative abundance of proteobacteria depended greatly on the composition of the residues, with a gradient observed from alfalfa to wheat, suggesting that this bacterial group may represent a good indicator of crop residues degradability and modifications during the decomposition process.

µgreen-db: a reference database for the 23S rRNA gene of eukaryotic plastids and cyanobacteria.

Studying the ecology of photosynthetic microeukaryotes and prokaryotic cyanobacterial communities requires molecular tools to complement morphological observations. These tools rely on specific genetic markers and require the development of specialised databases to achieve taxonomic assignment. We set up a reference database, called µgreen-db, for the 23S rRNA gene. The sequences were retrieved from generalist (NCBI, SILVA) or Comparative RNA Web (CRW) databases, in addition to a more original approach involving recursive BLAST searches to obtain the best possible sequence recovery. At present, µgreen-db includes 2,326 23S rRNA sequences belonging to both eukaryotes and prokaryotes encompassing 442 unique genera and 736 species of photosynthetic microeukaryotes, cyanobacteria and non-vascular land plants based on the NCBI and AlgaeBase taxonomy. When PR2/SILVA taxonomy is used instead, µgreen-db contains 2,217 sequences (399 unique genera and 696 unique species). Using µgreen-db, we were able to assign 96% of the sequences of the V domain of the 23S rRNA gene obtained by metabarcoding after amplification from soil DNA at the genus level, highlighting good coverage of the database. µgreen-db is accessible at http://microgreen-23sdatabase.ea.inra.fr.

Reduced microbial diversity induces larger volatile organic compound emissions from soils.

Microorganisms in soil are known to be a source and a sink of volatile organic compounds (VOCs). The role of the microbial VOCs on soil ecosystem regulation has been increasingly demonstrated in the recent years. Nevertheless, little is known about the influence of the microbial soil community structure and diversity on VOC emissions. This novel study analyzed the effect of reduced microbial diversity in soil on VOC emissions. We found that reduced levels of microbial diversity in soil increased VOC emissions from soils, while the number of different VOCs emitted decreased. Furthermore, we found that Proteobacteria, Bacteroidetes and fungi phyla were positively correlated to VOC emissions, and other prokaryotic phyla were either negatively correlated or very slightly positively correlated to VOCs emissions. Our interpretation is that Proteobacteria, Bacteroidetes and fungi were VOC producers while the other prokaryotic phyla were consumers. Finally, we discussed the possible role of VOCs as mediators of microbial interactions in soil.

Tillage intensity and pasture in rotation effectively shape soil microbial communities at a landscape scale.

Soil microorganisms are essential to agroecosystem functioning and services. Yet, we still lack information on which farming practices can effectively shape the soil microbial communities. The aim of this study was to identify the farming practices, which are most effective at positively or negatively modifying bacterial and fungal diversity while considering the soil environmental variation at a landscape scale. A long-term research study catchment (12 km2 ) representative of intensive mixed farming (livestock and crop) in Western Europe was investigated using a regular grid for soil sampling (n = 186). Farming systems on this landscape scale were described in terms of crop rotation, use of fertilizer, soil tillage, pesticides treatments, and liming. Molecular microbial biomass was estimated by soil DNA recovery. Bacterial and fungal communities were analyzed by 16S and 18S rRNA gene pyrosequencing. Microbial biomass was significantly stimulated by the presence of pasture during the crop rotation since temporary and permanent pastures, as compared to annual crops, increased the soil microbial biomass by +23% and +93% respectively. While soil properties (mainly pH) explained much of the variation in bacterial diversity, soil tillage seemed to be the most influential among the farming practices. A 2.4% increase in bacterial richness was observed along our gradient of soil tillage intensity. In contrast, farming practices were the predominant drivers of fungal diversity, which was mainly determined by the presence of pastures during the crop rotation. Compared to annual crops, temporary and permanent pastures increased soil fungal richness by +10% and +14.5%, respectively. Altogether, our landscape-scale investigation allows the identification of farming practices that can effectively shape the soil microbial abundance and diversity, with the goal to improve agricultural soil management and soil ecological integrity.

Response of soil bacterial community structure to successive perturbations of different types and intensities.

In soil, genetic structure modifications of indigenous bacterial community consecutively to a severe stress (mercury contamination) were delayed when the community was pre-exposed to various minor perturbations (heat, copper and atrazine). Such minor perturbations induced transitory community structure modifications leading to an increase of community stability towards a severe mercury stress. These results illustrated well the short-term pre-adaptation process for bacterial community hypothesizing that community submitted to perturbations become more resistant to withstand another stress.

Protein and DNA fingerprinting of a soil bacterial community inoculated into three different sterile soils.

The functional and genetic structures of a soil bacterial community were characterized after inoculation into three different sterile soils using a protein and DNA fingerprinting method, respectively. Principal component analysis (PCA) of profiles revealed that, depending on soil characteristics, bacterial communities with similar genetic structures harbored different functional structures and thus could potentially be of differing ecological significance for soil functioning. Co-inertia analysis between protein fingerprinting data and the corresponding sets of soil physicochemical characteristics demonstrated the correlation between the functional structure of the bacterial community and soil parameters, with pH, clay and CaCO(3) contents being the most discriminating factors.

Profiles of volatile organic compound emissions from soils amended with organic waste products.

Volatile Organic Compounds (VOCs) are reactive compounds essential to atmospheric chemistry. They are mainly emitted by living organisms, and mostly by plants. Soil microbes also contribute to emissions of VOCs. However, these emissions have not yet been characterised in terms of quality and quantity. Furthermore, long-term organic matter amendments are known to affect the microbial content of soils, and hence the quantity and quality of VOC emissions. This study investigates which and how much of these VOCs are emitted from soil amended with organic waste products (OWPs). Four OWPs were investigated: municipal solid waste compost (MSW), green waste and sludge co-compost (GWS), bio-waste compost (BIOW) and farmyard manure (FYM). These OWPs have been amended every two years since 1998 until now at a rate of ~4 tC ha-1. A soil receiving no organic inputs was used as a reference (CN). VOCs emissions were measured under laboratory conditions using a Proton Transfer Reaction-Quadrupole ion guide Time of Flight-Mass Spectrometry (PTR-QiToF-MS). A laboratory system was set up made of two Pyrex chambers, one for samples and the second empty, to be used as a blank. Our results showed that total VOC emissions were higher in BIOW than in MSW. Further findings outlined that the most emitted compounds were acetone, butanone and acetaldehyde in all treatments, suggesting a common production mechanism for these compounds, meaning they were not affected by the OWP amendment. We isolated 21 VOCs that had statistically different emissions between the treatments and could therefore be considered as good markers of soil biological functioning. Our results suggest that organic matter and pH jointly influenced total VOC emissions. In conclusion, OWPs in soil affect the type of VOC emissions and the total flux also depends on the pH of the soil and the quantity of organic matter.

Soil microbial diversity drives the priming effect along climate gradients: a case study in Madagascar.

The priming effect in soil is proposed to be generated by two distinct mechanisms: 'stoichiometric decomposition' and/or 'nutrient mining' theories. Each mechanism has its own dynamics, involves its own microbial actors, and targets different soil organic matter (SOM) pools. The present study aims to evaluate how climatic parameters drive the intensity of each priming effect generation mechanism via the modification of soil microbial and physicochemical properties. Soils were sampled in the center of Madagascar, along climatic gradients designed to distinguish temperature from rainfall effects. Abiotic and biotic soil descriptors were characterized including bacterial and fungal phylogenetic composition. Potential organic matter mineralization and PE were assessed 7 and 42 days after the beginning of incubation with 13C-enriched wheat straw. Both priming mechanisms were mainly driven by the mean annual temperature but in opposite directions. The priming effect generated by stoichiometric decomposition was fostered under colder climates, because of soil enrichment in less developed organic matter, as well as in fast-growing populations. Conversely, the priming effect generated by nutrient mining was enhanced under warmer climates, probably because of the lack of competition between slow-growing populations mining SOM and fast-growing populations for the energy-rich residue entering the soil. Our study leads to hypotheses about the consequences of climate change on both PE generation mechanisms and associated consequences on soil carbon sequestration.

Biofilm biodiversity in French and Swiss show caves using the metabarcoding approach: First data.

In recent decades, show caves have begun to suffer from microorganism proliferation due to artificial lighting installations for touristic activity. In addition to the aesthetic problem, light encourages microorganisms that are responsible for physical and chemical degradation of limestone walls, speleothems and prehistoric paintings of cultural value. Microorganisms have previously been described by microscopy or culture-dependent methods, but data provided by new generation sequencing are rare. The authors identified, for the first time, microorganisms proliferating in one Swiss and in four French show caves using three different primers. The results showed that both photosynthetic and non-photosynthetic bacteria were the dominant taxa present in biofilms. Microalgae were heavily represented by the Trebouxiophyceae, Eustigmatophyceae and Chlorophyceae groups. Twelve diatoms were also recorded, with dominance of Syntrichia sp. (96.1%). Fungi were predominantly represented by Ascomycota, Zygomycota and Basidiomycota, fully half of the sampled biofilms where Fungi were detected. Comparing microbial communities from bleach-treated caves to those in untreated caves showed no significant difference except for a low-level change in the abundance of certain taxa. These findings provided by Illumina sequencing reveal a complex community structure in the 5 caves based on the assembly of bacteria, cyanobacteria, algae, diatoms, fungi and mosses.