Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives.

Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on possible solutions involving the use of nanomaterials to ensuring the biosensors' selectivity. Overall, the examples presented illustrate the various tools available when developing enzyme biosensors for new applications and stress the necessity to more comprehensively investigate their selectivity and validate the biosensors versus standard analytical methods.

Flow injection enzymatic biosensor for aldehydes based on a Meldola Blue-Ni complex electrochemical mediator.

Carbon nanofibers (CNF) are efficient electrode modifiers in electrochemical biosensors that enhance the electrochemical active area, induce electrocatalytic effect toward the oxidation of the enzymatic cofactor nicotinamide adenine dinucleotide (reduced form, NADH), and enable the quantitative immobilization of enzymes. Combining CNF with efficient and stable mediators radically augments the speed of electron transfer between NADH and solid electrodes and leads to electrochemical sensors characterized by high sensitivity and stability. The main aim of this work was to investigate the performance of a novel mediator for NADH with advantageously low solubility in an electrochemical detector based on a screen-printed CNF electrode as well as its potential in biosensing. Using a mediator, prepared from Meldola Blue and Ni hexamine chloride, a stable and sensitive electrochemical NADH sensor is provided with a detection limit of 0.5 µmol L-1. Further on, covalent immobilization of a recently described aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 strain on the surface of the mediator-modified electrode produced a stable biosensor for the detection of aldehydes. When integrated in a flow injection analysis (FIA) setup with amperometric detection at 0.1 V vs. Ag/AgCl, the measurement of benzaldehyde with a detection limit of 10 µmol L-1 over a linear range of 30-300 µmol L-1 is possible. Determination of trace benzaldehyde impurities in a pharmaceutical excipient was also demonstrated and results compared with a chromatographic method. Graphical abstract.

Dynamics of Necrophagous Insect and Tissue Bacteria for Postmortem Interval Estimation During the Warm Season in Romania.

The estimation of postmortem interval (PMI) is affected by several factors including the cause of death, the place where the body lay after death, and the weather conditions during decomposition. Given the climatic differences among biogeographic locations, the understanding of necrophagous insect species biology and ecology is required when estimating PMI. The current experimental model was developed in Romania during the warm season in an outdoor location. The aim of the study was to identify the necrophagous insect species diversity and dynamics, and to detect the bacterial species present during decomposition in order to determine if their presence or incidence timing could be useful to estimate PMI. The decomposition process of domestic swine carcasses was monitored throughout a 14-wk period (10 July-10 October 2013), along with a daily record of meteorological parameters. The chronological succession of necrophagous entomofauna comprised nine Diptera species, with the dominant presence of Chrysomya albiceps (Wiedemann 1819) (Calliphoridae), while only two Coleoptera species were identified, Dermestes undulatus (L. 1758) and Creophilus maxillosus Brahm 1970. The bacterial diversity and dynamics from the mouth and rectum tissues, and third-instar dipteran larvae were identified using denaturing gradient gel electrophoresis analysis and sequencing of bacterial 16S rRNA gene fragments. Throughout the decomposition process, two main bacterial chronological groups were differentiated, represented by Firmicutes and Gammaproteobacteria. Twenty-six taxa from the rectal cavity and 22 from the mouth cavity were identified, with the dominant phylum in both these cavities corresponding to Firmicutes. The present data strengthen the postmortem entomological and microbial information for the warm season in this temperate-continental area, as well as the role of microbes in carcass decomposition.

Extremozyme-Based Biosensors for Environmental Pollution Monitoring: Recent Developments.

Extremozymes combine high specificity and sensitivity with the ability to withstand extreme operational conditions. This work presents an overview of extremozymes that show potential for environmental monitoring devices and outlines the latest advances in biosensors utilizing these unique molecules. The characteristics of various extremozymes described so far are presented, underlining their stability and operational conditions that make them attractive for biosensing. The biosensor design is discussed based on the detection of photosynthesis-inhibiting herbicides as a case study. Several biosensors for the detection of pesticides, heavy metals, and phenols are presented in more detail to highlight interesting substrate specificity, applications or immobilization methods. Compared to mesophilic enzymes, the integration of extremozymes in biosensors faces additional challenges related to lower availability and high production costs. The use of extremozymes in biosensing does not parallel their success in industrial applications. In recent years, the "collection" of recognition elements was enriched by extremozymes with interesting selectivity and by thermostable chimeras. The perspectives for biosensor development are exciting, considering also the progress in genetic editing for the oriented immobilization of enzymes, efficient folding, and better electron transport. Stability, production costs and immobilization at sensing interfaces must be improved to encourage wider applications of extremozymes in biosensors.

Draft genome sequence data of Antarctic Penicillium sp. strain E22, from Deception Island.

Here, we report the draft genome sequence and assembly of the Penicillium sp. strain E22, which was isolated from Antarctic soil of Deception Island, South Shetland Islands close to the Antarctic Peninsula. The genome was sequenced using a 2 # 250 bp paired-end method by Illumina MiSeq 6000. The genome assembly was performed using softwares implemented in the Kbase web service. The phylogenetic tree of strain E22 comparing its internal transcribed spacer (ITS) region with the other Penicillium showed high genetic similarity to Penicillium griseofulvum MN545450 and Penicillium camemberti MT530220. Draf genome of Penicillium sp. strain E22 comprises 33,653 coding sequences, with a high G + C content of 48.32% and a total size of 37,484,944 bp. This draft genome assembly version has been deposited at GenBank under accession JASJUN000000000.

Bioprospecting for Novel Bacterial Sources of Hydrolytic Enzymes and Antimicrobials in the Romanian Littoral Zone of the Black Sea.

Marine microorganisms have evolved a large variety of metabolites and biochemical processes, providing great opportunities for biotechnologies. In the search for new hydrolytic enzymes and antimicrobial compounds with enhanced characteristics, the current study explored the diversity of cultured and uncultured marine bacteria in Black Sea water from two locations along the Romanian coastline. Microbial cell density in the investigated samples varied between 65 and 12.7 × 103 CFU·mL-1. The total bacterial community identified by Illumina sequencing of 16S rRNA gene comprised 185 genera belonging to 46 classes, mainly Gammaproteobacteria, Alphaproteobacteria, Flavobacteriia, and 24 phyla. The 66 bacterial strains isolated on seawater-based culture media belonged to 33 genera and showed variable growth temperatures, growth rates, and salt tolerance. A great fraction of these strains, including Pseudoalteromonas and Flavobacterium species, produced extracellular proteases, lipases, and carbohydrases, while two strains belonging to the genera Aquimarina and Streptomyces exhibited antimicrobial activity against human pathogenic bacteria. This study led to a broader view on the diversity of microbial communities in the Black Sea, and provided new marine strains with hydrolytic and antimicrobial capabilities that may be exploited in industrial and pharmaceutical applications.

First report on antibiotic resistance and antimicrobial activity of bacterial isolates from 13,000-year old cave ice core.

Despite the unique physiology and metabolic pathways of microbiomes from cold environments providing key evolutionary insights and promising leads for discovering new bioactive compounds, cultivable bacteria entrapped in perennial ice from caves remained a largely unexplored life system. In this context, we obtained and characterized bacterial strains from 13,000-years old ice core of Scarisoara Ice Cave, providing first isolates from perennial ice accumulated in caves since Late Glacial, and first culture-based evidences of bacterial resistome and antimicrobial compounds production. The 68 bacterial isolates belonged to 4 phyla, 34 genera and 56 species, with 17 strains representing putative new taxa. The Gram-negative cave bacteria (Proteobacteria and Bacteroidetes) were more resistant to the great majority of antibiotic classes than the Gram-positive ones (Actinobacteria, Firmicutes). More than 50% of the strains exhibited high resistance to 17 classes of antibiotics. Some of the isolates inhibited the growth of clinically important Gram-positive and Gram-negative resistant strains and revealed metabolic features with applicative potential. The current report on bacterial strains from millennia-old cave ice revealed promising candidates for studying the evolution of environmental resistome and for obtaining new active biomolecules for fighting the antibiotics crisis, and valuable cold-active biocatalysts.

Microbiome pattern of Lucilia sericata (Meigen) (Diptera: Calliphoridae) and feeding substrate in the presence of the foodborne pathogen Salmonella enterica.

The microbial diversity and quantitative dynamics during the insect's development stages constitute recently developed putative tools in forensic and medical studies. Meanwhile, little is known on the role of insects in spreading foodborne pathogenic bacteria and on the impact of these pathogens on the overall insects and feeding substrate microbiome composition. Here, we provide the first characterization of the bacterial communities harbored in adult and immature stages of Lucilia sericata, one of the first colonizers of decomposed human remains, in the presence of the foodborne pathogen Salmonella enterica using 16S rRNA Illumina sequencing and qPCR. The pathogen transmission from the wild adults to the second generation was observed, with a 101.25× quantitative increase. The microbial patterns from both insect and liver samples were not influenced by the artificial introduction of this pathogenic foodborne bacteria, being dominated by Firmicutes and Proteobacteria. Overall, our results provided a first detailed overview of the insect and decomposed substrate microbiome in the presence of a human pathogen, advancing the knowledge on the role of microbes as postmortem interval estimators and the transmission of pathogenic bacteria.

Heat Shock Response of the Active Microbiome From Perennial Cave Ice.

Ice caves constitute the newly investigated frozen and secluded model habitats for evaluating the resilience of ice-entrapped microbiomes in response to climate changes. This survey identified the total and active prokaryotic and eukaryotic communities from millennium-old ice accumulated in Scarisoara cave (Romania) using Illumina shotgun sequencing of the ribosomal RNA (rRNA) and messenger RNA (mRNA)-based functional analysis of the metatranscriptome. Also, the response of active microbiome to heat shock treatment mimicking the environmental shift during ice melting was evaluated at both the taxonomic and metabolic levels. The putatively active microbial community was dominated by bacterial taxa belonging to Proteobacteria and Bacteroidetes, which are highly resilient to thermal variations, while the scarcely present archaea belonging to Methanomicrobia was majorly affected by heat shock. Among eukaryotes, the fungal rRNA community was shared between the resilient Chytridiomycota and Blastocladiomycota, and the more sensitive Ascomycota and Basidiomycota taxa. A complex microeukaryotic community highly represented by Tardigrada and Rotifera (Metazoa), Ciliophora and Cercozoa (Protozoa), and Chlorophyta (Plantae) was evidenced for the first time in this habitat. This community showed a quick reaction to heat shock, followed by a partial recovery after prolonged incubation at 4°C due to possible predation processes on the prokaryotic cluster. Analysis of mRNA differential gene expression revealed the presence of an active microbiome in the perennial ice from the Scarisoara cave and associated molecular mechanisms for coping with temperature variations by the upregulation of genes involved in enzyme recovery, energy storage, carbon and nitrogen regulation, and cell motility. This first report on the active microbiome embedded in perennial ice from caves and its response to temperature stress provided a glimpse into the impact of glaciers melting and the resilience mechanisms in this habitat, contributing to the knowledge on the functional role of active microbes in frozen environments and their response to climatic changes.

Draft Genome Sequence of Flavobacterium sp. Strain PL002, Isolated from Antarctic Porphyra Algae.

Here, we report the draft genome sequence of Flavobacterium sp. strain PL002, isolated from Antarctic Porphyra algae. The 4,299,965-bp genome sequence is assembled into 170 contigs, has 32.92% GC content, and 3,734 predicted genes.

Draft Genome Sequence of Antarctic Psychrotroph Streptomyces fildesensis Strain INACH3013, Isolated from King George Island Soil.

The draft genome sequence of Streptomyces fildesensis strain INACH3013, a psychrotrophic bacterium isolated from Northwest Antarctic soil, was reported. The genome sequence totaling 9,306,785 bp resulted from 122 contigs characterized by a GC content of 70.55%.

Potential bacterial biomarkers for insect colonization in forensic cases: preliminary quantitative data on Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica dynamics.

For the last decades, forensic microbiology became an emerging complementary tool in criminalistics. Although the insect-microbe interactions regarding pathogen transmission were extensively studied, only scarce information is available on bacterial transfer from necrophagous insects to host tissues. Our data provides the first report on the occurrence of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica in Lucilia illustris Meigen, 1826 (Diptera: Calliphoridae), and the quantitative dynamics of the two bacterial species along the insect life-stages and transfer to beef and pork host tissues using qPCR gyrase b specific primers. The content of both bacterial species increased along the insect life stages. W. chitiniclastica was detected in all developmental stages independent of the feeding substrate. I. indica was measurable with 102 gene copies ng-1 DNA threshold starting from the third instar larvae when feeding on beef, and from the egg stage with a 102× higher representation when using the pork substrate. The transfer of bacterial species to both tissues occurred after 3 colonization days except for I. indica that was visible in beef liver only during day 5. Considering the utilization of pork tissues as human analogues, these quantitative microbial dynamics data provides first insect-specific bacterial candidates as potential colonization biomarkers in forensic investigations.

Advances in the Detection of Dithiocarbamate Fungicides: Opportunities for Biosensors.

Dithiocarbamate fungicides (DTFs) are widely used to control various fungal diseases in crops and ornamental plants. Maximum residual limits in the order of ppb-ppm are currently imposed by legislation to prevent toxicity problems associated with excessive use of DTFs. The specific analytical determination of DTFs is complicated by their low solubility in water and organic solvents. This review summarizes the current analytical procedures used for the analysis of DTF, including chromatography, spectroscopy, and sensor-based methods and discusses the challenges related to selectivity, sensitivity, and sample preparation. Biosensors based on enzymatic inhibition demonstrated potential as analytical tools for DTFs and warrant further research, considering novel enzymes from extremophilic sources. Meanwhile, Raman spectroscopy and various sensors appear very promising, provided the selectivity issues are solved.

Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.

In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

Exploring the hydrolytic potential of cultured halophilic bacteria isolated from the Atacama Desert.

Considering that most industrial processes are carried out under harsh physicochemical conditions, which would inactivate enzymes from commonly isolated mesophilic organisms, current studies are geared toward the identification of extremophilic microorganisms producing enzymes resistant to extreme salt concentrations, temperature and pH. Among the extremophiles, halophilic microorganisms are an important source of salt-tolerant enzymes that can be used in varying biotechnological applications. In this context, the aim of the present work was to isolate and identify halophiles producing hydrolases from the Atacama Desert, one of the harshest environments on Earth. Isolates were recovered from halite samples and screened for the presence of seven different hydrolase activities (amylase, caseinase, gelatinase, lipase, pectinase, cellulase and inulinase) using agar plate-based assays. From a total of 23 halophilic bacterial isolates, most showed lipolytic (19 strains) and pectinolytic (11 strains) activities. The molecular identification of eight selected isolates showed a strong similarity to members of the Halomonas and Idiomarina genera. Therefore, the present study represents a preliminary, but essential, step to identify novel biological sources of extremozymes in an environment once thought to be devoid of life.

Total and Potentially Active Bacterial Communities Entrapped in a Late Glacial Through Holocene Ice Core From Scarisoara Ice Cave, Romania.

Our understanding of the icy-habitat microbiome is likely limited by a lack of reliable data on microorganisms inhabiting underground ice that has accumulated inside caves. To characterize how environmental variation impacts cave ice microbial community structure, we determined the composition of total and potentially active bacterial communities along a 13,000-year-old ice core from Scarisoara cave (Romania) through 16S rRNA gene Illumina sequencing. An average of 2,546 prokaryotic gDNA operational taxonomic units (OTUs) and 585 cDNA OTUs were identified across the perennial cave ice block and analyzed in relation to the geochemical composition of ice layers. The total microbial community and the putative active fraction displayed dissimilar taxa profiles. The ice-contained microbiome was dominated by Actinobacteria with a variable representation of Proteobacteria, while the putative active microbial community was equally shared between Proteobacteria and Firmicutes. Accordingly, a major presence of Cryobacterium, Lysinomonas, Pedobacter, and Aeromicrobium phylotypes homologous to psychrotrophic and psychrophilic bacteria from various cold environments were noted in the total community, while the prevalent putative active bacteria belonged to Clostridium, Pseudomonas, Janthinobacterium, Stenotrophomonas, and Massilia genera. Variation in the microbial cell density of ice strata with the dissolved organic carbon (DOC) content and the strong correlation of DOC and silicon concentrations revealed a major impact of depositional processes on microbial abundance throughout the ice block. Post-depositional processes appeared to occur mostly during the 4,000-7,000 years BP interval. A major bacterial composition shift was observed in 4,500-5,000-year-old ice, leading to a high representation of Beta- and Deltaproteobacteria in the potentially active community in response to the increased concentrations of DOC and major chemical elements. Estimated metabolic rates suggested the presence of a viable microbial community within the cave ice block, characterized by a maintenance metabolism in most strata and growth capacity in those ice deposits with high microbial abundance and DOC content. This first survey of microbial distribution in perennial cave ice formed since the Last Glacial period revealed a complex potentially active community, highlighting major shifts in community composition associated with geochemical changes that took place during climatic events that occurred about 5,000 years ago, with putative formation of photosynthetic biofilms.

Temperature Influence on Prevailing Necrophagous Diptera and Bacterial Taxa With Forensic Implications for Postmortem Interval Estimation: A Review.

The decomposition process of human (and other mammalian) remains is influenced by numerous factors such as the environmental temperature, relative humidity, precipitation, geographical location of the remains, as well as the medical conditions of and any injuries sustained by the deceased person. The decomposition process generally follows a consistent pattern (fresh, bloat, active decomposition, advanced decomposition, and dry stage). Understanding the time-span of each stage of decomposition and the influence of the biotic and abiotic factors involved is imperative when trying to estimate the time elapsed since death (postmortem interval [PMI]). Over the course of decomposition, the tissues are gradually consumed by necrophagous insects and bacteria. The environmental temperature and its variations influence how insects colonize the remains, having a significant impact on their presence and developmental cycle. Additionally, the bacterial community colonizing decomposing tissues is also greatly affected by variations in environmental temperature. Because both the rate of decomposition of human remains and the relative abundance of certain insects and bacterial species are all temperature-dependent, insect and bacterial colonization data are useful as key points for the PMI estimation. This article reviews the current literature documenting the data collected on the occurrence and development cycles of predominant necrophagous Diptera (Calliphoridae, Muscidae, Sarcophagidae) and bacterial taxa involved in the decomposition of various carcasses at different temperatures. This review will impact the forensic community by providing an overview on the temperature, insect and bacterial records for the PMI estimation, seeking to aid forensic entomologists, microbiologists, pathologists, and the legal community.

Characterization and microbial analysis of first recorded observation of Conicera similis Haliday (Diptera: Phoridae) in forensic decomposition study in Romania.

The identification of necrophagous insect diversity and dynamics has forensic significance for postmortem interval estimation specific to burial. Few studies regarding the necrophagous entomofauna from buried remains have been performed to date. In contrast to the exposed carcasses, the accessibility of soil to insects is limited due to burial depth and is dependent on soil type. This study highlights the colonization behavior of Conicera similis (Haliday 1833) (Diptera: Phoridae) during carcass decomposition, a previously unobserved taxon in Romania. Adult and larvae specimens were collected from rat (Rattus norvegicus) carcasses buried at 40 cm depth in an urban park environment in June 2016 during active decomposition and their presence and activity period was correlated with the environmental parameters variation. Bacterial diversity from C. similis female adult and larvae specimens was determined via 16S rRNA gene Illumina sequencing to further characterize these commonly encountered and forensically important necrophagous insects. This report signals the easternmost geographical location in Europe (Bucharest, Romania) of C. similis to date.

Characterizing forensically important insect and microbial community colonization patterns in buried remains.

During violent criminal actions in which the perpetrator disposes of the victim's remains by burial, the analysis of insects and bacterial colonization patterns could be necessary for postmortem interval (PMI) estimation. Our research aimed to assess the decomposition process of buried rat carcasses from shallow graves (40 cm), the diversity and dynamics of insects and bacteria throughout the decomposition stages, and the environmental parameters' influence on these variations. The results provide further insight on decomposition in soil and contribute to a broader understanding of the factors involved in decomposition by qualitatively and quantitatively analysing the decomposer community (bacteria and insects). Additionally, two bacterial taxa, Enterococcus faecalis and Clostridium paraputrificum that were investigated for the first time as PMI indicators using quantitative polymerase chain reaction (qPCR) showed differential abundance over time, promising data for PMI estimation. The current study on the decomposition of buried rat carcasses in a natural environment will strengthen the current knowledge on decomposed remains from shallow graves and represents an effort to quantify insect and bacterial taxa as PMI estimators.

Bacterial and archaeal community structures in perennial cave ice.

Ice entrenched microcosm represents a vast reservoir of novel species and a proxy for past climate reconstitution. Among glacial ecosystems, ice caves represent one of the scarcely investigated frozen habitats. To characterize the microbial diversity of perennial ice from karst ecosystems, Roche 454 sequencing of 16S rRNA gene amplicons from the underground ice block of Scarisoara Ice Cave (Romania) was applied. The temporal distribution of bacterial and archaeal community structures from newly formed, 400, and 900 years old ice layers was surveyed and analyzed in relation with the age and geochemical composition of the ice substrate. The microbial content of cave ice layers varied from 3.3 104 up to 7.5 105 cells mL-1, with 59-78% viability. Pyrosequencing generated 273,102 reads for the five triplicate ice samples, which corresponded to 3,464 operational taxonomic units (OTUs). The distribution of the bacterial phyla in the perennial cave ice varied with age, organic content, and light exposure. Proteobacteria dominated the 1 and 900 years old organic rich ice deposits, while Actinobacteria was mostly found in 900 years old ice strata, and Firmicutes was best represented in 400 years old ice. Cyanobacteria and Chlorobi representatives were identified mainly from the ice block surface samples exposed to sunlight. Archaea was observed only in older ice strata, with a high incidence of Crenarchaeota and Thaumarchaeaota in the 400 years old ice, while Euryarchaeota dominated the 900 years old ice layers, with Methanomicrobia representing the predominant taxa. A large percentage (55.7%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxa levels, suggesting a greater undiscovered bacterial diversity in this glacial underground habitat. The prokaryotes distribution across the cave ice block revealed the presence of 99 phylotypes specific for different ice layers, in addition to the shared microbial community. Ice geochemistry represented an important factor that explained the microbial taxa distribution in the cave ice block, while the total organic carbon content had a direct impact on the cell density of the ice microcosm. Both bacterial and archaeal community structures appeared to be affected by climate variations during the ice formation, highlighting the cave ice microbiome as a source of putative paleoclimatic biomarkers. This report constitutes the first high-throughput sequencing study of the cave ice microbiome and its distribution across the perennial underground glacier of an alpine ice cave.