https://dx.doi.org/10.21577/0103-5053.

20230148

Review J. Braz. Chem. Soc. 2024, 35, 1, e-20230148, 1-16

©2024 Sociedade Brasileira de Química

The World of Cave Microbiomes: Biodiversity, Ecological Interactions, Chemistry,

and the Multi-Omics Integration

Natália N. Kato,a Gabriel S. Arini,a Ricardo Roberto Silva,a Maria Elina Bichuette,b
José Augusto P. Bitencourtc and Norberto P. Lopes *,a
a
Departamento de Ciência Biomoleculares, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, 14040-900 Ribeirão Preto-SP, Brazil
b
Laboratório de Estudos Subterrâneos, Departamento de Ecologia e Biologia Evolutiva,
Universidade Federal de São Carlos, 13565-905 São Carlos-SP, Brazil
Instituto Tecnológico Vale, 66055-090 Belém-PA, Brazil
c

Caves are ecosystems and natural heritage sites with magnificent biological diversity, from
microorganisms to animals, despite their selective environment for development and survival. Until
now, studies on cave microbiomes have included taxonomic classification through metagenomics
analysis, demonstrating microbiological heterogeneity mainly composed of prokaryotic organisms
or bacteria species. The generally oligotrophic environment, with limited energy input and dark
zone, is the main feature differentiating the cave microorganisms from the other microbiomes
and resulting in a unique habitat, which has unexplored regard to biology and chemical diversity.
Multi-omics integration is a key strategy for scanning and integrating the macro and micromolecular
universe, providing a better comprehension of processes and answers of the microbiome in natural
cavities. Here, we highlight the strategies employed to study the cave microbiome, from the
microbial genomes to their ability to secondary metabolites production, and also the multi-omics
integration to explore the chemical and biological diversity in caves.

Keywords: caves, metagenomics, metabolomics, prokaryotes, metabolites, multi-omics

1. Introduction absence of light are the main characteristics of a cave

extreme habitat to a living organism.7 Conversely, recent
The cave ecosystem provides rough environmental research shows that some subterranean habitats may
conditions for microbial survival;1 however, the substrates not be limited by the energy input5 since their substrate
from the caves host heterogeneous microbiota with have a high availability of organic matter through floods
ecological properties. 2,3 Even more, some studies and animal excreta (e.g., bat guano). Autochthonous
highlighted the importance of the microbial community microorganisms from caves have metabolic strategies
as key organisms at the base of the subterranean food web enabling survival in the highly selective environment for
and in decomposition processes.4,5 The microbial groups life, hence may be a source of unprecedented microbial
most relevant in caves are the Fungi kingdom, Archaea, bioproducts.
and Bacteria domain, the last one is more representative The integration of the system of biology provides
and widely reported in the literature. different ecological answers to living organisms in the
Once the cave physicochemical properties and cave environment. Likewise, the cave microbiome may
environmental variables act on the ecosystem biodiversity, be a potential source for novel organic compounds,8
the trophic levels or the light intensity are relevant to especially bacterial species, which are already known
determining the division inside a cave and the distribution for secondary metabolite production. For this reason, the
of microorganisms in the substrates.6 The oligotrophy and multi-omics integration (genome, transcriptome, proteome,
and metabolome) is a strategy to achieve new insights and
*e-mail: [email protected] perspectives about the biology and chemistry of the cave
Editor handled this article: Paulo Cezar Vieira ecosystem. In this perspective, our group has been working

This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Kato et al. The World of Caves Microbiomes

with different omics models of ecological integration 2. Cave Microbiome Diversity

of microorganisms in the environment or among other
species9 and the isolation of metabolites produced by the Environmental variables and trophic conditions
endophytic microorganism.10 Recently, we related for the contribute to the uniqueness of the habitat and shape
first time the occurrence of a symbiosis mechanism between the diversity and abundance of the microbiota in an
amphibians and microorganisms.11,12 Within the framework ecosystem. Regarding the metabolism requirement, the
of cellular adhesion, our study combines the strategy using caves microorganisms express functional enzymes as
different omics techniques and biological assay, which a driving force for catalyzing reactions using minerals
allow us to highlight the inhibition of adhesion mechanisms, or inorganic matter to survive in low nutrient input.18
decreasing bacterial colonization.13 Chemolithoautotrophic microorganisms oxidize inorganic
Also, we evaluated the chemistry of marine molecules such as iron,19 manganese,20,21 ammonia,22,23
microorganisms,14 combining the transcriptomics and methane,24,25 and sulfur26,27 which are the most abundant
metabolomics approaches to comprehend the interactions in caves substrates. The metabolic trait of these organisms
between marine organisms and their microbiota,15 also the varies according to the inorganic matter availability and
quencher effects of singlet oxygen species by microalgae.16 the ecological role in the ecosystem as the biogeochemical
In advanced technologies, we develop images to evaluate process, 28,29 which is involved in a cave’s physical
the interactions between plants and microorganisms.17 structuration maintenance.
Regarding the ecological interactions of microbiomes Caves as a sink of methane favor the high diversity
in the environment, we began the metabolomics and of methanotrophic microorganisms. The ability of the
transcriptomics studies of microorganisms living in methanotrophic species to survive and develop in poor
substrates with high concentrations of iron ore and dark nutritional substrate consists of its preference to use
zones in Brazilian caves. carbon from methane molecules as an oxidative energy
This literature review brings an overview of the Bacteria source.30 The major microbial groups in caves are the
diversity associated with the cave ecosystem. As well as the methanotrophs belonging to the Archaea and Bacteria, as
high-throughput genomic sequencing to the identification α- and γ-proteobacteria species,31,32 shown in Figure 1. Both
of the microbial taxonomic profile and ecological functions microbial groups have a significant part of unculturable
in the caves. On the other hand, metabolomics brings to species thus, metagenomic sequencing determines the
light microbiome interactions as well as the opportunity taxonomic profile and the genes expressing enzymes with
to unveil novel metabolic pathways. Other important ecological functions in caves.
aspects, like characteristics and variables of the caves, are The nitrogen cycle involves essential reactions mediated
correlated with the diversity, abundance, and distribution by the microorganisms, catalyzing a series of reactions in
of microorganisms in substrates. At last, the integration which nitrogen from N2 atmospheric and ammonia are
of advanced tools may improve our comprehension of the fixated through its oxidized form as NO2− and NO3− in the
cave microbiome. subterranean habitats.22 In the oligotrophic environment,

Figure 1. Bacterial phylogenetic profile in caves, the correlation between the culture-independent (violet) and dependent (orange), both using targeted
and untargeted metagenomics analysis.

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The World of Cave Microbiomes Kato et al.

there are biological and ecological markers common in soil through deoxyribonucleic acid (DNA) and ribonucleic
and sediment, represented by the specific microbial species acid (RNA) sequences to distinguish among bacteria,
with nitrogen-fixing and ammonia-oxidizing capacity,33 archaea, fungi, and algae. The overwhelming majority of
i.e., they use inorganic nitrogen for the development of living microorganisms in an ecosystem are uncultivable
extremophilic microorganisms.34-36 The metagenomic in controlled conditions, somehow affecting the exact
analysis using caves samples emphasizes the presence taxonomic classification. 38 Thus, the high-throughput
of these microorganisms by the functional enzymes of sequencing in the genomic field has advanced a significative
nitrogen cycle metabolism (Table 1). step to bring to light the microbial “dark matter” and is a
Heterotroph microorganisms in caves represent some breakthrough in cave microbiome studies.
bacterial groups and the fungi kingdom, which profit Genomic sequencing involves two strategies using
from the high organic matter biomass in the substrates, environmental DNA fragments: untargeted and targeted
e.g., guano, using organic carbon as an energy source.37 analysis. The shotgun is an untargeted technique that
In the underground system, the heterotrophic species are identifies a complex microbial sample without choosing
found in the entrance and twilight zones or according a specific set of microorganisms. 39 The targeted or
to the trophic levels schematized by Ghosh et al.,6 also metabarcoding analysis focuses on the conservative regions
confirming the relevance of division into the cave for of ribosomal RNA, making a more selective sequencing
microbiome studies or the distinction of microbial groups. method to determine taxonomic microbial domains. 40
Heterotrophic metabolism requires high organic input and Besides that, the taxonomic profile classification requires
more mechanisms to sustain the metabolic reactions, such a representative database to compare genomic sequences,
as inorganic sources or light, making them more selective. which is still growing to deposit new genetic sequences of
In an underground system with extremophile organisms, microbial strains.
uncultivable protocols are employed for the determination Metagenomics distinguishes each domain by DNA
of widespread microorganisms in nature, nurturing in extraction of all living organisms in the environmental
different mechanisms and energy sources. Next-generation samples to gene sequencing.41 There are many targets
sequencing (NGS) technology is a high-throughput way to of conserved regions of ribosomal RNA utilized in
identify microorganism taxonomy and differentiate living environmental metabarcoding studies, but the majority of
organisms, and genes prediction involved in ecological rRNA used in the classification of microbial domains at
functions in a low-cost and fast procedure. genus and species levels are 16S-23S rRNA to prokaryotes42
and 18S rRNA to eukaryotes.43 And, for fungal DNA
3. Molecular Methodologies to Microbial barcoding is sequenced the internal transcribed spacer (ITS)
Taxonomic Classification in Caves regions,44 revealing the fungi diversity in the environment.
However, we focused on the methodologies using RNA
The microbial “dark matter” challenges the awareness sequencing involving: DNA extraction from samples,
of all genomes of living organisms composing an followed by polymerase chain reaction (PCR) amplification
ecosystem. Over the years, NGS methodologies enabled with good-quality DNA, and the comparison between
the barcode of the native microbiome constituents hypervariable regions of ribosomal RNA and the genomic

Table 1. Enzymes and genes correlated with microbial ecological functions in caves

Gene functional marker Enzyme Predictive functions in cave ecosystem

pmoA- amoA, pmoB- amoB, pmoC-amoC, mmoX,
mmoY, mmoZ, mmoB, mmoC, mmoD, mxaF/xoxF, methane monooxygenase (MMO) oxidation of methane for metabolic reactions
gmaS, mauA, mcrA,
nitrogen cycle metabolism
pmoA- amoA, pmoB- amoB, pmoC-amoC
ammonia assimilation
anfG, vnfD, vnfE, vnfK, vnfG, vnfH, nifH, nifD,
nitrogen cycle assimilation
and nifK
RuBisCO CO2 fixation
ATP-citrate lyase carbon assimilation
sulfide-quinone reductase (SQR) and Sox system sulfide oxidation
Quorum sensing antimicrobial resistance
ATP: adenosine triphosphate.

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Kato et al. The World of Caves Microbiomes

database.38 The ribosomal RNA is most used in samples microbial identification in sub-species level resolution
directly collected from caves to analyze the biodiversity in without the cultivation and isolation steps and the functional
the niche and is applied to the identification of the isolated genes shown in Table 1.48 The drawbacks of the shotgun
strain. analysis are the requirement of a large genomic databank,
The identification of bacteria in caves is successfully high-biomass samples, and expensive costs.39 Besides, the
processed by the molecular systematic using 16S rRNA library has a limited cover of environmental samples but
or 23S rRNA genomic sequencing45 through culture- is better for pathogenic microorganisms and the human
dependent and independent protocols.46 The 16S rRNA microbiome.48
gene sequencing presents high coverage of bacterial groups The main difference between 16S rRNA and shotgun
at the genus and species level in environmental samples metagenomic sequencing is the resolution of the taxonomic
widely used in caves phylogenetic investigations in culture- level in the bacterial community; shotgun provides
dependent and independent protocols.47 Figures 1 and 2 species-strain level data while the 16S rRNA covers genus
show the comparison of bacterial richness using different and species.49 The advantage of shotgun metagenomic
methodologies. These data were obtained through the sequencing over the 16S rRNA analysis, and other
original articles contained in the “Clarivate Analytics Web metabarcoding targets, is the determination of the
of Science”, covering from 2010 to 2023 and the keywords ecological functions through the specific genes without
used to research were “cave microorganisms” and “cave any specific microbial target.34,50
microbiomes”.
Even the most common phyla in caves-the 4. The Bacterial Community in the Ecosystem
Actinomycetes-reveals that three different substrates of the Caves
analyzed by both techniques show a decrease in the
variability of cultivation methods.46 In this way, reinforcing Prokaryotic cells are highly adapted to inhospitable
the effectiveness of the culture-independent by offering a environments through biochemical reactions enabling
magnificent overview of free-living organisms widespread survival despite the adversities herein, bacteria domain
in the cave environment and the hugeness of unculturable-or is widely distributed in oligotrophic substrates and
unknown-microorganisms to encourage biological studies. aphotic zone in caves. The main groups described in the
On the other hand, the cultivated-based methods of bacterial cave ecosystem are Actinobacteria, α-Proteobacteria,
species are essential to understanding the entire microbial γ-Proteobacteria, β-Proteobacteria, δ-Proteobacteria,
genome and biology on high throughput and also as a Bacteriodetes, Acidobacteria, Firmicutes, and others in
requirement for chemical investigation. smaller proportion, shown in Figure 1. Cave features and
factors shape the diversity and abundance of bacterial
groups, which is related to their genotype, and the phenotype
implies metabolic responses.29 In this way, microbial
metabolism is essential to determine the development of
particular bacterial species and the ecological interactions,
despite the selective environment for life.
The Actinobacteria phylum is ubiquitously and well-
adapted to several environmental variables or adversities,
besides that, it is a filamentous bacteria which have
ecological importance in nature.51 Buresova-Faitova et al.52
reported autochthonous actinobacterial species in
pristine caves, evidencing that the intact and conservative
Figure 2. Correlation among techniques to Bacteria domain identification niche is capable of hosting new bacterial strains. Rare
in cavities microbiomes, using Next-Generation sequencing. 16S CI microorganisms in caves are indicators of undiscovered
refers to the microorganisms identified by the culture-independent
method; 16S CD for culture-dependent, and the shotgun for untargeted genomes to expand the knowledge about the biodiversity
metagenomics. in the poorly exploited ecosystem.
The metabolic pathways prediction, through the NGS
Shotgun metagenomics is an untargeted analysis to methods, indicates that actinobacterial species participate
identification of the metagenomes of all living organisms in ecologic functions in the niche, e.g., the biogeochemical
from an ecosystem, such as bacteria, viruses, fungi, and processes53 and energy flow equilibrium.50 These metabolic
protozoa. Cave substrates presented a high efficacy to features make the actinobacteria the most relevant group

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The World of Cave Microbiomes Kato et al.

in the underground ecosystem; besides that, there is the biofilm formation and adhesion to the surface, like in
biotechnological and pharmacological interest due to walls and rocks, sustained by an extracellular polymeric
their extensive secondary metabolism. The Streptomyces matrix.64 The biofilm is a bacterial association showing
and Nocardia species are present in the unfavorable a high degree of communication among the cells via
environment of caves, which are isolated for chemical quorum sensing.49,65 Due to the high humidity of the cave
studies signaling biological properties in different targets.54 environment, the biofilm is sampled to identify the bacterial
The second largest phyla in caves, α-, β-, δ-, and taxonomic diversity and the ecological interaction among
γ-Proteobacteria, are Gram-negative bacteria, and it is other specimens in the microbial network.
related to being a possible biomarker in the extremophilic The synergism of microorganisms is critical to promote
environment because of their metabolism. Autotrophic adaptation in oligotrophic conditions and darkness.
Proteobacteria are widespread on sediment, water, and rock Therefore, the co-existence of some bacterial species in the
in caves28,55 since their metabolism enables them to thrive in community is related to the presence of others, predicted
unfavorable conditions.56 The methanogenic proteobacteria by the functional genes responsible for both metabolic
oxidize methane as a sole carbon and energy source cooperations.18,28 The interactions among microorganisms
from substrates in caves.57 Thus, the cave microbiome in any ecological niche are challenging in controlled
contributes to the high diversity of proteobacteria species, conditions. Also, the cultivation protocols commonly
but there are no studies about the bioproducts, and the involve monoculture to isolate each strain, which may
cultivation protocols still challenge to deepen the chemical override the existence of other bacteria species. In an
investigations. extreme environment, such as the caves, the obtention of
The heterotrophs bacteria in caves need high organic these microorganisms is even harder.
matter content, and many of them are pathogenic for
humans and may be emerging new diseases. The substrates 5. How do Environmental Variables Influence
with high amounts of organic content host heterotrophic the Cave Microbiome?
species belonging to the Firmicutes phylum,58 which
also may be an outcome of local anthropization with The environmental variables affecting the cave
the introduction of waste disposal or human microbiota. ecosystem are divided into two groups: the biotic and
Otherwise, the Firmicutes species, in scarcity of nutrients, abiotic factors. The biotic factors are related to the
use strategies to get energy by interacting with other interactions among living organisms, such as fauna,
microorganisms, thus, the result of this mechanism is the flora, and microbiota, or biological interactions. The
identification of enzymes related to antibiotic resistance.59,60 abiotic factors concern the temperature, humidity, pH,
Despite the pathogenic properties, Bacillus licheniformis or geological composition, which means the physical
isolated from caves provide peptides with antibacterial and properties,66,67 besides the localization and seasons.68,69 And
antifungal effects,61 showing that Firmicutes species also then, each cave has particular characteristics to provide
produce bioactive compounds. specific conditions for microbiome development. These
In the entrance or twilight zone in caves, the cyanobacteria variations can, directly and indirectly, affect the diversity
species with photoautotrophic metabolism may develop,62 and abundance of microbiome constituents. In general,
but they require a high trophy level to survive, as well as microorganisms have features or acquire mechanisms in
the terrestrial microorganisms. In addition, groups found answer to the environmental properties of the cave.
in low proportions in caves substrates: Acidobacteria, The microorganisms are widespread on different
Bacteriodetes, Chloroflexi, and Planctomycetes are substrates such as sediment, guano, wall, rock, wood, water,
well-characterized by shotgun metagenomics and 16S soil, moonmilk, and others according to environmental
rRNA gene sequencing; on the other hand, there are conditions. The total organic carbon (TOC) parameter
limited data regarding their culturable bacteria species. measures the organic content from the substrate, which
Reinforcing that the taxonomic diversity and abundance enables the correlation with the taxonomic profile and
of the microorganisms are correlated strongly with the their metabolic responses.70-72 The availability of organic
environmental variables. matter offers an appropriate requirement for heterotroph
The interactions among microorganisms are favorable or chemoorganotroph microorganisms’ development.28 In
to their survival in any niche, as in the cave substrates. other words, the energy input defines the microbial biomass
Heterotroph species need interaction with autotroph groups and composition given the metabolic demand.
to provide energy and survive in scarcity conditions.63 In The human presence and the fauna habituating caves,
other cases, the complex interspecies interaction leads to mainly bats, deposit organic content in this ecosystem,

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Kato et al. The World of Caves Microbiomes

stimulating the development of selective microbial groups. The biological and physical parameters are mensurable
Guano is an excellent substrate for heterotrophic microbial in caves and are possible to correlate with NGS techniques to
proliferation, which also recycles the organic content identify taxonomy patterns distributed in the ecosystem.28,84
in the substrate73 despite reports about pathogenicity of Despite the ecological contributions of the microbial “dark
guano and promotion of high proliferation of pathogenic matter” remaining unknown, Figure 3 summarizes the
microorganisms.37 Otherwise, the presence of animals is correlation between cave environmental properties, such as
essential to the biogeochemical process and the equilibrium the incidence of light and trophic levels, in the microbiome
of the environment. and their ecological interactions in nature.
The anthropization process affects the native microbial
composition with the insertion of human microbiota,
resulting in decreased local biodiversity. 74,75 These
microorganisms can modify the cave microbiome,
which may interact negatively and cause geological
deterioration.18,76 For this, preservative measures are
necessary to avoid the degradation of cave structures or
even the loss of genetic heritage.77 The human intervention
modifies the pristine cave microbiome for the presence of
pathogenic microorganisms.
Regarding the physical aspects of the ecosystem of the
caves, the atmospheric gases inside caves, usually composed
of O2, CO2, NO2, N2O, and NH3, play a crucial role as an
inorganic energy source.22 As a requirement of microbial
metabolism, they catch specific gases to obtain inorganic Figure 3. Schematic view inside a cave according to the three zones: the
carbon, nitrogen, and oxygen as a starter for each metabolic photic (high incidence of light); the dysphotic (transition between the
entrance and the dark); and the aphotic (absence of light). (a) Entrance,
cycle. The composition and concentration of atmospheric outside view; (b) entrance, inside view; (c) and (d) fauna hosting in caves
gases influence the variability of microorganisms along ecosystem; (e) deep zone.
the cave and reinforce their importance for ecosystem
maintenance. 6. Ecological Functions of Cave Microbiome
The pH variability in substrates influences the
distribution of microorganisms, ranging by the microbial The microbiota is omnipresent in ecosystems, but
affinity to acid or alkaline substrate.78 Acidic caves favor each microorganism composing the environment has its
the development of specific microbial strains in low-pH complexity and dynamic, challenging the comprehension of
substrates. This ability is related to the genetic modifications the microbial network in an ecosystem.85 Microbial enzymes
of specific strains belonging to the same genus and directly catalyze reactions to microbiome maintenance, interactions
influences the microbial distribution inside caves.26 These among the organisms, or response to environmental
modifications affect microbial metabolism, showing variations,50 of which are predicted using NGS approaches.
differences among the populations according to the The ecological functions of the microbiome in caves depend
capacity to resist different pH conditions.71 The mineral on the genotype and phenotype of each microorganism,
precipitations, such as CaCO3, by the microbiome in caves, influencing their transcriptome, proteome, and metabolome.
depending on the metabolism and pH.53,79 The influences of environmental variables in caves
Whether the samples are collected in sediment microorganisms are evidenced by the mechanisms of
from a wall, ceiling, or rock, there is another parameter resistance to antibiotics enzyme-mediated, even in locations
commonly measured in a cave environment reflecting preserved from the anthropization process. 86 These
the composition and biomass. 72,80,81 Mineralogical mechanisms of cave-dwelling microorganisms to survive in
composition varies according to the cave formation and the environment reflect their antibiotic resistance, even when
directly influences the microbial composition due to the compared to terrestrial ones.59 Enzymatic metabolic changes
availability of substrate to the metabolic cycle.82,83 The of microbial population in caves are related to competitive
concentration and variability of minerals may illustrate interaction among microorganisms in oligotrophic substrates,
the distribution of microorganisms along the cave and which also justify their ecological role in the niche.
their capacity to catalyze reactions and get the energy to The genomic analysis and bioinformatic tools allow the
survive in cave substrates. prediction of enzymes with ecological importance in nature,

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The World of Cave Microbiomes Kato et al.

which is widely applied in ubiquitous microorganisms.28 Several pathways and mechanisms correlate to
The functions of bacteria widely discussed and established microbe adaptation and survival as a dynamic response
in natural cavities are geochemical reactions19,81,87 and of microorganisms in caves, either by quorum sensing
atmospheric gas transformation. 22,88 Most pathways genes as a competition among microorganisms, and
prediction in the caves microbiome focused on energy an interesting side of these events is the formation of
metabolism or mechanism of defense in competition bioproducts. Microbial metabolism uses organic and
with other microbial groups to assure survival in extreme inorganic matter to incorporate the components in their
conditions.7,89 Table 1 lists specific functional genes biomass through functional genes and specific enzymes,70
and enzymes responsible for the maintenance of cave which also is favorable to studies of the chemical diversity
ecosystems identified by DNA sequencing in metagenomics in this ecosystem.
analysis. The production of complex metabolites is still untapped
Key metabolic genes (pmoA, mmoX, mxaF/xoxF, due to the limitation of culture-dependent methodologies
and gmaS) belonging to methane oxidation, with pmoA to procedure the chemical analysis. The main bottleneck
and mmoX are mainly involved in coding methane is the environmental conditions supplied to microorganism
monooxygenase (MMO) family enzymes, catalyzing development in oligotrophic substrates. Metagenomics
methane from the environment in order to get energy.56 advances have led to determining the taxonomy and their
The pmoA enzyme is a functional biomarker widely genes capable of producing high-complexity compounds:
described in methanotrophic microorganisms, able to e.g., Polyketide Synthase (PKS) and Nonribosomal
oxidize the methane from the environment. Over the years, Peptide Synthetases (NRPS) pathways.6,95,96 Although the
the modulation of the physical structuration of the caves is chemical analyses of cave-dwelling bacteria are limited,
linked to the methanotrophic activity, being relevant to the the NGS and transcriptomics research on underground
ecological role in the ecosystem. ecosystems indicates metabolic pathways to the production
The Bacteria and Archaea domains biologically of secondary metabolites.
mediate the reactions of the nitrogen cycle along the cave,
and the abundance and diversity depend on the atmospheric 7. Chemical Studies and Biological Properties
N2 availability.90 The main functional biomarkers, amoA of the Isolated Microorganisms
and nifDKH genes, indicate the expression of enzymes
capable of converting atmospheric nitrogen into ammonia Natural products are the primary source of bioactive
or nitrogen fixed to profit the living organisms in the cave metabolites or a scaffold to develop new drugs with
substrate. In extreme environmental conditions, such as regulatory agencies approval and widely commercialized.97,98
oligotrophy, lack of light, and pH variations, the nitrogen As mentioned before, environmental factors and occurrence
availability in the substrate is the main source of nutrition influence the microbial phenotype, and cultivation
to the microbiome,33 justifying the prevalence of the protocols enable the evaluation of metabolite production
ammonia-oxidizing and nitrogen-oxidizing biomarkers in in reason of their metabolism, such as the PKS and NRPS
cave ecosystem. pathways.96 The occurrence of new compounds in the
The vnf and anf genes are “alternative” nitrogenases cave-dwelling microorganisms leads to the investigation of
in Bacteria, helping in the fixation of N2 in environments how microorganisms living in the inhospitable conditions
with an absence of molybdenum (Mo), but the presence of caves and isolated from substrates far from their origin
of vanadium (V; vnf) or iron (Fe; anf).91 Mo is the main (e.g., soil and marine environment) may influence the
cofactor of nif gene and a scarcer and limiting micronutrient biosynthetic pathway to the production of secondary
in terrestrial environments.92 Although both nitrogenases metabolites.
are structurally similar to nif produced, separate gene As an example of other ecosystems, terrestrial or surface
clusters encode the vnf and anf. Also, vnf and anf have environments started with the bacteria as a source for the
different catalytic properties and perform a more efficient discovery of bioactive compounds, in which the species
conversion of N2 in higher temperatures.92,93 Although nif from Actinobacteria are the common group producer of
genes are the predominate form in nature, it is common antibiotics molecules, using a sensing strategy to survive
to find microorganisms that have more than one type of with a complex of microorganisms. The marine microbiome
nitrogenase, such as Anabaena variabilis, a heterotrophic, is widely explored for the isolation of bacteria to investigate
heterocyst-forming and nitrogen-fixing cyanobacterium (nif the promissory source of compounds. Also, the hypogean
and vnf) Nostoc spp.94 and some other diazotrophs such and inhospitable ecosystem contribute to the research of
Azotobacter vinelandii (nif, vnf, and anf).91 new microbial genotypes.

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Kato et al. The World of Caves Microbiomes

As an innovative source, the cave’s extreme substrates to sub-inhibitory concentrations of antibiotics, and metal
host bacteria species able to be cultivated for evaluation supplementation, are strategies to explore their chemical
of secondary metabolites with biotechnological and diversity.101 Thus, actinobacterial species were susceptible
pharmacological properties.8,99 Ghosh et al.6 report the to different stimuli in the growing medium that influence
antibacterial activity from Actinobacteria, Proteobacteria, the biosynthetic pathways.
and Firmicutes and summarize the biological effects of Table 2 and Figure 4 list the secondary metabolites from
another microbial domain. On this wise, the oligotrophic cave Actinobacteria according to their biological activity.
and aphotic environment entails strategies to thrive in As well known, antibiotic metabolites have been reported
caves, as the inter-species interactions commit their in soil-terrestrial bacteria as the inter-species competition;
metabolic pathway to enzyme expression and metabolites in this way, specific species produce enzymes belonging to
biosynthesis. the PKS and NRPS, which in cultivation protocols activate
As the most predominant and abundant phyla in the the biosynthesis of secondary metabolites.96 Besides the
cave ecosystem, the Actinobacteria species are targets in antimicrobial compounds, the cytotoxic effects are related
studies involving bioactive compounds and are considered a to the naphthoquinone derivatives against colon (TCT-1
source of new antibiotics.100 The cave-derived actinomycete and Caco-2), lung (A549), ovary (SKOV3), and cervix
produces high-complexity compounds with biological (Hela) cancer cell lines.102-104 Researching new scaffolds
activity and varies according to the culture media and for cancer treatment is very challenging, and the extreme
methodology.54 The response of Actinobacteria strains environment may host distinct microorganisms as a source
under different stimuli, such as co-cultivation, exposure of bioactive compounds.

Table 2. Secondary metabolites isolated from caves microorganisms

Substrate Taxonomic classification Identified compound Biological activity Reference

Bacteria domain
Soil Streptomyces sp. undecylprodigiosin (1) antioxidant 105
hypogeamicin A (2)
hypogeamicin B (3) cytotoxic to colon cell line
Soil Nonomuraea sp. 106
hypogeamicin C (4) (TCT-1)
hypogeamicin D (5)
cytotoxic to tumoral cell lines
(A549, MCF-7, HepG-2, HeLa,
Soil Streptomyces sp. xiakemycin A (6) 104
HCT-116, SH-SY5Y, and PC-3)
and antibiotic
cyclodysidin D
Moonmilk Streptomyces sp. 107
chaxalactin B
huanglongmycin A (7) antibiotic and cytotoxic to
Soil Streptomyces sp. huanglongmycin B (8) tumoral cell lines (A549, 103
huanglongmycin C (9) SKOV3, Hela, and Caco-2)
Micromonospora sp. aloesaponarin II (10)
Nonomuraea sp. hypogeamicin B (3)
Sediment 101
Microbispora sp. tetarimycin B (11)
Streptosporangium sp. funisamine
diketopiperazines (12), (13), (14)
Streptomyces sp. ICC1
nordentatin (15)
Sediment antimicrobial activity 108
2’, 5’-dimethoxyflavone (16)
Streptomyces sp. ICC4
diazepinomicin
4,10-dichloroanthrabenzoxocinone (17)
10,12-dichloroanthrabenzoxocinone (18)
4,12-dichloroanthrabenzoxocinone (19)
Streptomyces isolates M2_9, antibacterial, antifungal, and
Water, moonmilk 4,10-dichloro-3-O- 109
M4_24, and M5_8 anticancer activities
methylanthrabenzoxocinone (20)
10,12-dichloro-3-O-
methylanthrabenzoxocinone (21)
Streptomyces lunaelactis
Moonmilk lunaemycin A and derivatives antibacterial 110
MM109T

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The World of Cave Microbiomes Kato et al.

Figure 4. Selected secondary metabolites produced by cave microorganisms. Table 2 contains each compound associated with the bacterial species;
substrates, where the microorganisms were isolated, and their biological effects.

The first step to the prospection of bioactive compounds when there is a microbial target, the selective medium
in caves microbiome depends on the cultivated-based is employed to prevail the growth of a specific bacteria
techniques, focusing on the best choice for isolation and group, which is applied to studies of Streptomyces genera
fermentation in selective culture medium. The cultivation using ISP2 culture medium, a yeast malt extract medium.111
of cave microorganisms and sampling are challenging The strategies to get a high diversity of microorganisms
and vary according to growth metabolism and culture from environmental samples are variations in the culture
medium composition. The slow-growing species requires medium compositions, the incubation time optimization,
poor culture media, such as the R2A (Reasoner’s 2A agar) temperature, and rotation in shaker equipment. All these
culture medium,102 since the fast-growing species may parameters are essential to reach a good quality of isolated
suppress its development in the cultivation. In contrast, microorganisms in chemical investigations.
microorganisms with rapid metabolism grow in a medium After the isolation and fermentation of microorganisms
with a high concentration of nutrients.104 In other cases, in the liquid medium, the secondary metabolites are

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Kato et al. The World of Caves Microbiomes

extracted from the supernatant through different conditions, biodiversity and their ecological function in the hypogean
such as the liquid-liquid partition between an organic ecosystem.
solvent, e.g., ethyl acetate, obtaining the medium Transcriptomic science enables the assessment of
polarity compounds,103,106 or by drying the supernatant active genes in microbial cells correlating the functions in
and suspension in methanol. 111 The next step is the a particular ecosystem via high-throughput methods. NGS
determination of the composition of the extracts obtained for transcriptome studies provides valuable data about each
from isolated strains. gene responsible for biological reactions in the metabolic
Aiming the knowledge of the high diversity of cycle of a set of organisms in the ecosystem at a given time
metabolites produced by cave microorganisms, mass point.116 Moreover, these active genes are responsible for
spectrometry allows us the annotation compounds based expressing enzymes to biomolecule production, like the
on spectral data or fragmentation patterns.112,113 For the PKS and NRPS pathways to the biosynthesis of secondary
metabolomics data acquisition, the ionization step is metabolites.95,96 Table 2 shows the functional genes and the
crucial for the detection of analytes, thus, electrospray corresponding enzymes of their metabolic cycle.
ionization is widely employed for low molecular weight Proteomics evaluates a set of proteins of the
organic compounds detection,114 which in turn is highly organisms and correlates them with metabolic functions
suitable for the characterization of metabolites produced and maintenance of cells. Specific genes are responsible
by microorganisms. The spectrometry data are processed for transcript expression to produce intracellular
for the annotation of compounds or discovery of new biomolecules.117,118 The proteomic analysis identifies
metabolites, comparing in the platform, as the Global a microbial strain based on its structural proteins or
Natural Products Social Molecular Networking (GNPS).112 intrinsic metabolites. 119 In the caves, the application
Lastly, the metabolomics advancements and statistical of mass spectrometry using matrix-assisted laser
analysis promote an overview of the microbiome, desorption/ionization time of flight (MALDI-TOF)
distinguishing or grouping microorganisms conforming for protein characterization provided the taxonomic
to their metabolomes in different biological and ecological profile of airborne biomass to compare with the tourism
contexts. season, reporting the impact of human intervention
on the shape of the natural microbiome. These
8. The Omics Sciences in the Underground variables assessment with proteomics studies give more
Ecosystem information about biodiversity distribution in caves
ecosystem, metabolic flux, and ecological function.120
Omics sciences allow the measurement and analysis of The genomic, transcriptomic, and proteomic unravel the
biological structure, involving micro to macromolecular ecosystem particularities or interactions in the microbial
analysis, or from genotypes to phenotypes, of a specific community.
individual in the population. Here, we highlighted The untapped underground ecosystem converges to
the biology system for comprehension of a particular research for autochthonous microorganism and their
microbiome, which consists of the four main areas of chemical and biological potential in an entire ecosystem.
the omics field: genomic, transcriptomic, proteomic, and Top-down strategies show the response of the cave
metabolomic. Environmental features affect different microbiome under environmental stressors and the
degrees in the system of biology. The multi-omics influence on chemical diversity. The chemical profile data
integration provides an overview of a whole ecosystem, enable the distinction of microbiome constituents, outlier
even in particular conditions, resulting in comprehension identification, and prediction of the relevant chemical
improvement of the biology in the extreme microbiome.115 markers related to environmental variables. The research of
A high-throughput DNA sequencing approach bioactive metabolites in caves microbiome may be assessed
provides taxonomic classification and functional genomes. by the metabolomics statistical analysis and avoid isolation
Metagenomics successfully determines the entire genome steps of each compound.121
of the microbial population, even the uncultivated ones.39,45 Metabolomics methodologies are employed in natural
These analyses may provide the taxonomic classification of products research, bringing different answers about the
caves microorganisms and their functional genes involving metabolism.122 In this regard, metabolomics is a magnificent
the metabolic cycle.50 The section about methodologies approach to correlating environmental factors with
for microbial identification, using culture-dependent and microbial metabolome based on the chemical profile of
independent, reveals the importance of metagenomics to each individual in the microbiome.123 The response of cave
evaluate the impact of microbial cave groups on the local microorganisms was observed through the biosynthesis

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The World of Cave Microbiomes Kato et al.

of secondary metabolites according to the exposure to Of the possible ways of performing integration, multi-
stressor stimuli in the cultivable protocols, in which the staged analysis and meta-dimensional analysis are the
multivariate statistical analysis pointed out the specific two main approaches to data integration (Figure 5).129
metabolites produced by different conditions.101,124 The Multi-staged analysis basically integrates a dataset from
cave conditions shape the microbiome biodiversity and a sequential or hierarchical approach. That is, in a linear
metabolism; therefore, they can provide unprecedented or hierarchical way, in a sequence of steps, the analysis
metabolites or new occurrences for drug discovery and offer is divided into several axes where two different scales
valuable insights considering the ecology relationships. inscribed in a given time are used to build an explanatory
The omics sciences are the way to understand the model. In such a way, the association between the
knowledge gap of the microbial community associated different types of data is first sought and, then, the result
with the cave environment.125 The integration of data of this association with the phenomenon of interest.
multi-omics explores a set of statistical strategies to get Meta-dimensional analysis is based on the multiple
information on a specific ecosystem and its effects on the integration of different types of data simultaneously,
organisms.126 The multi-omics integration is the pathway to building a multivariate model associated with a given
explore in different biological levels the living organisms result. That is, constructing an explanatory model is
of the microbiome in natural cavities, also may be the tool based on combining results from different scales and
for discovering new genomes and bringing to light the merging them. This type of approach, in turn, can be
challenging world of microbial “dark matter”. divided into three different strategies: (i) concatenation-
based integration, which involves the concatenation of
9. Multi-Omics Integration as a Way to all omic data obtained in a given experimental set into a
Illuminate the Cave Bacteria Biology single matrix; (ii) transformation-based integration, where
appropriate transformations are applied to the data in
One of the great challenges when dealing with such a way that they lead to a uniform set of information,
non-model microorganisms, especially in the case of allowing the combination into merged datasets; (iii)
extremophile specimens such as bacteria living in caves, model-based integration, where machine learning models
relies on how to access them from a biological point of are obtained separately for each dataset before the actual
view and unravel the molecular potential contained in their combination.129-131
secondary metabolism. The identification of this molecular
framework is one of the current bottlenecks in this context.127
In this sense, a systemic approach offers the possibility of
considering a new model of study from a holistic point of
view. In other words, this type of approach considers the
complex interrelationship between the different degrees of
organization at the molecular level, offering a solid basis
for explaining certain traits observed. The combination of
different data sources, especially those obtained on a large
scale, such as genomics, transcriptomics, proteomics, and
metabolomics, has been used in technological advances
and the discovery of new molecules, their respective
metabolic pathways of origin, and ecological interactions.9
The increase in the availability of this type of data is one
of the main reasons for the cheapening of omics analyses,
especially regarding sequencing techniques and, gradually,
the use of mass spectrometry.113 Of the advantages of Figure 5. The integration of omics approaches illuminates different
using this type of integrative multi-omic approach, in aspects of the biology of the cave microorganisms.
addition to holistic considerations on the study organism,
Chauvel et al.128 also highlight the decreased risks of false The integration of omics data has shown an outstanding
positives, since different data sources point in the same potential in the examination of molecular complex
direction and the possibility of new insights regarding the underpinnings.132 Maansson et al.,127 using an integrative
relationship between molecular interplay and its phenotypic approach where they combined metabolomics and
response for a given trait. genomics data obtained from 13 species closely related

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Kato et al. The World of Caves Microbiomes

to Pseudoalteromonas luteoviolacea, discovered new genes and their ecological role. The integration of genomic,
antimicrobial bioactive compounds belonging to the transcriptomic, proteomic, and metabolomic is a potential
thiomarinols class, which until now had not been described, strategy to fulfill and overcome the knowledge gap about
as well as the identification of the biosynthetic gene cave microbiomes.
cluster for indolmycin, which traditional methods could
not predict. In addition to the discovery of new bioactive Acknowledgments
compounds, as well as their biosynthetic gene clusters, data
integration is also a good strategy for the study of microbial The authors thank the funding agencies Fundação
interaction.133,134 Rieusset et al.135 in order to assess the Amparo à Pesquisa do Estado de São Paulo (FAPESP,
impact of the environment on the secondary metabolism 2022/0578-0) and Coordenação de Aperfeiçoamento de
of Pseudomonas spp., using cross-data metabolomics Pessoal de Nível Superior (CAPES) for financial support,
integration, were able to show that the production of several and Pâmela Rodrigues Rosa Pereira, PhD student, for
secondary metabolites produced by Pseudomonas spp. figures elaboration.
were significantly modulated when such bacteria were
cultivated when in contact with plant roots, where this Author Contributions
difference favored root growth. Despite such advances
in the use of multi-omics approaches in the study of new Natalia N. Kato was responsible for conceptualization, data curation,
models, it is noted that a significant part of the papers writing the original draft review and editing; Gabriel S. Arini for data
focuses on two sets of omics data, generally opting for curation and writing the original draft, review and editing; Ricardo
the integration between genomic and metabolomics data.9 R. Silva for data curation and writing review and editing; Maria E.
In this sense, it would be interesting not only to use other Bichuette data curation, writing the original draft, review and editing;
omics data platforms but also to integrate them in order to José A. P. Bitencourt conceptualization, writing the original draft,
present more robust and reliable models for the biological review and editing; and Norberto P. Lopes conceptualization, writing
phenomenon of interest. This is because, as was very well the original draft, review and editing.
presented by Zaramela et al.134 genomics presents what is
possible to be happening, transcriptomics presents what Natália Naomi Kato obtained a
is possibly happening, proteomics what makes a given BSc and MSc degree in Pharmaceutical
process happen and, finally, metabolomics presents what Sciences at the Universidade Federal
is actually happening. de Mato Grosso do Sul. Her thesis
research involves the chemistry of
10. Conclusions cave-dwelling bacteria using mass
spectrometry and metabolomics studies.
Since the crescent advances in science and the
challenges for the treatment of diseases, new strategies Gabriel S. Arini received his BSc
in hard-to-reach environments and integration of omics degree in Pharmaceutical Sciences and
sciences contribute to investigations of new metabolites Chemistry at the Faculdades Oswaldo
biologically active. The exploration of caves microbiome Cruz and MSc in Biological Sciences
is the source of rare microorganisms or microbial at the Universidade de São Paulo. He
phenotypes correlated to the environment adversities works with computational biology,
leading to metabolites discovery. The advances in bioinformatics, mass spectrometry, and metabolomics.
metagenomic analysis led to taxonomic classification in
cave ecosystems and correlation with the active functional Maria Elina Bichuette is Professor
genes. However, the microbial richness in caves still in the Universidade Federal de São
represents unexplored biodiversity, and it is necessary Carlos. She graduated in Biological
to improve the genomic field to increase the knowledge Sciences at the Universidade de São
about possibly extremophile microorganisms. Paulo. She got the MSc and PhD
There are some bottlenecks to the comprehension of degree in Zoology at the Universidade
caves microorganisms, and the integration of multi-omics de São Paulo. She works in the fauna of subterranean
provides new insights into caves studies. The investigation habitats, such as the caves.
of microorganisms in extreme conditions successfully
started with the metagenomic approach to characterize

12 of 16 J. Braz. Chem. Soc. 2024, 35, 1, e-20230148

The World of Cave Microbiomes Kato et al.

Ricardo Roberto da Silva obtained 7. Zgonik, V.; Mulec, J.; Eleršek, T.; Ogrinc, N.; Jamnik, P.; Ulrih,
a BSc in Forest Engineer and MSc in N. P.; Microorganisms 2021, 9, 2326. [Crossref]
Genetics at the Universidade Federal 8. Zada, S.; Sajjad, W.; Rafiq, M.; Ali, S.; Hu, Z.; Wang, H.; Cai,
de Viçosa and PhD degree in Biological R.; Microb. Ecol. 2022, 84, 676. [Crossref]
Sciences at the Universidade de 9. Brunetti, A. E.; Carnevale Neto, F.; Vera, M. C.; Taboada, C.;
São Paulo. He is Professor at the Pavarini, D. P.; Bauermeister, A.; Lopes, N. P.; Chem. Soc. Rev.
Department of BioMolecular Sciences, Universidade 2018, 47, 1574. [Crossref]
de São Paulo. His research aims to integrate different 10. Guimarães, D. O.; Borges, W. S.; Vieira, N. J.; de Oliveira, L.
data with computational statistics to the elucidation of F.; da Silva, C. H. T. P.; Lopes, N. P.; Dias, L. G.; Durán-Patrón,
metabolic pathways in different organisms, such as plants R.; Collado, I. G.; Pupo, M. T.; Phytochemistry 2010, 71, 1423.
and microorganisms. [Crossref]
11. Brunetti, A. E.; Lyra, M. L.; Melo, W. G. P.; Andrade, L. E.;
José Augusto Pires Bitencourt Palacios-Rodríguez, P.; Prado, B. M.; Haddad, C. F. B.; Pupo,
obtained a BSc in Biological M. T.; Lopes, N. P.; Proc. Natl. Acad. Sci. U. S. A. 2019, 116,
Sciences at the Universidade Federal 2124. [Crossref]
Fluminense, MSc and PhD degree in 12. Brunetti, A. E.; Bunk, B.; Lyra, M. L.; Fuzo, C. A.; Marani, M.
Marine Biology at the Universidade M.; Spröer, C.; Haddad, C. F. B.; Lopes, N. P.; Overmann, J.;
Federal Fluminense. Currently, he is ISME J. 2022, 16, 788. [Crossref]
Researcher at the Instituto Tecnológico Vale. His research 13. Trentin, D. S.; Silva, D. B.; Frasson, A. P.; Rzhepishevska, O.;
focuses on the microorganisms of Amazon canga and Da Silva, M. V.; De Pulcini, E. L.; James, G.; Soares, G. V.;
ferruginous cave using molecular approaches. Tasca, T.; Ramstedt, M.; Giordani, R. B.; Lopes, N. P.; Macedo,
A. J.; Sci. Rep. 2015, 5, 8287. [Crossref]
N o r b e r t o Pe p o r i n e L o p e s 14. Wilke, D. V.; Jimenez, P. C.; Branco, P. C.; Rezende-Teixeira,
is Full Professor at the School of P.; Trindade-Silva, A. E.; Bauermeister, A.; Lopes, N. P.; Costa-
Pharmaceutical Sciences of Ribeirão Lotufo, L. V.; Planta Med. 2021, 87, 49. [Crossref]
Preto at the Universidade de São 15. Costa-Lotufo, L. V.; Carnevale-Neto, F.; Trindade-Silva, A.
Paulo and Member of the Brazilian E.; Silva, R. R.; Silva, G. G. Z.; Wilke, D. V.; Pinto, F. C. L.;
Academy of Sciences. He obtained Sahm, B. D. B.; Jimenez, P. C.; Mendonça, J. N.; Lotufo, T.
a BSc and MSc degree in Pharmacy and PhD degree in M. C.; Pessoa, O. D. L.; Lopes, N. P.; Chem. Commun. 2018,
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