Shedding light on the composition of extreme microbial dark matter: alternative approaches for culturing extremophiles

More than 20,000 species of prokaryotes (less than 1% of the estimated number of Earth’s microbial species) have been described thus far. However, the vast majority of microbes that inhabit extreme environments remain uncultured and this group is termed “microbial dark matter.” Little is known regarding the ecological functions and biotechnological potential of these underexplored extremophiles, thus representing a vast untapped and uncharacterized biological resource. Advances in microbial cultivation approaches are key for a detailed and comprehensive characterization of the roles of these microbes in shaping the environment and, ultimately, for their biotechnological exploitation, such as for extremophile-derived bioproducts (extremozymes, secondary metabolites, CRISPR Cas systems, and pigments, among others), astrobiology, and space exploration. Additional efforts to enhance culturable diversity are required due to the challenges imposed by extreme culturing and plating conditions. In this review, we summarize methods and technologies used to recover the microbial diversity of extreme environments, while discussing the advantages and disadvantages associated with each of these approaches. Additionally, this review describes alternative culturing strategies to retrieve novel taxa with their unknown genes, metabolisms, and ecological roles, with the ultimate goal of increasing the yields of more efficient bio-based products. This review thus summarizes the strategies used to unveil the hidden diversity of the microbiome of extreme environments and discusses the directions for future studies of microbial dark matter and its potential applications in biotechnology and astrobiology

NOVOS MÉTODOS PARA ANÁLISE DA DIVERSIDADE MICROBIANA EM SISTEMAS DE TRATAMENTO DE RESÍDUOS SÓLIDOS E LÍQUIDOS

Population growth is accompanied by an alarming increase of solid and liquid waste production worldwide. The maintenance of soil and water reservoirs as well as their protection against pollution effects are of extreme importance for human civilization. Microorganisms are directly involved in biogeochemical cycles, being key drivers of the degradation of many carbon sources, as pollutants, which when properly known and managed provide a wide range of applications, including in wastewater, landfills and leachate treatment. Recent advancements in molecular tools to study the diversity and function of microbial communities are driving and contributing to a better understanding of microbial ecology, and researchers apply this knowledge to manage and understand biotechnological processes. In wastewater and leachate treatment, as well in landfills, microbial ecology techniques have been applied to study the behavior of microbial community during the process. This article will briefly outline the most widely used and the newest tools presenting their potential and limitations.El crecimiento de la población viene acompañado por el aumento alarmante de la producción de residuos sólidos y líquidos en todo el mundo. La manutención del suelo y de los cuerpos de agua y su protección contra los efectos de la contaminación son esenciales para la civilización humana. Los microorganismos están directamente relacionados con los ciclos biogeoquímicos, como agentes responsables de la degradación de varias fuentes de carbono, como numerosas sustancias contaminantes y, cuando son conocidos y manejados adecuadamente, proveen un amplio rango de aplicaciones biotecnológicas, incluyendo el tratamiento de aguas residuales, rellenos sanitarios y lixiviados. Avances recientes en el desarrollo de técnicas moleculares para el estudio de la diversidad y función de las comunidades microbianas están contribuyendo a un mejor entendimiento de la ecología microbiana y los investigadores aplican este conocimiento para entender, desarrollar y perfeccionar procesos biotecnológicos. Estas técnicas de ecología microbiana son aplicadas para estudiar la estructura y el perfil de las comunidades microbianas durante el proceso de tratamiento de aguas residuales y lixiviados, así como en rellenos sanitarios. El presente trabajo documenta brevemente las herramientas más utilizadas actualmente para el estudio de comunidades microbianas en estos ambientes, y presenta algunas nuevas y prometedoras técnicas, con sus aplicaciones potenciales y limitaciones. O crescimento cada vez maior da população mundial é acompanhado pelo aumento alarmante da produção e deposição de resíduos sólidos e líquidos no mundo inteiro. A manutenção de reservatórios de água e de solos, assim como a proteção desse ambientes contra os efeitos da poluição, são essenciais para a manutenção da civilização humana. Os microrganismos estão diretamente envolvidos nos ciclos biogeoquímicos sendo os responsáveis pela degradação de inúmeras fontes de carbono, como muitos poluentes e, quando devidamente conhecidos e manejados, podem ser utilizados em uma vasta gama de aplicações biotecnológicas, incluindo tratamento de esgotos, aterros sanitários e lixiviados. Avanços recentes no desenvolvimento de técnicas moleculares utilizadas no estudo da diversidade e função de comunidades microbianas vêm possibilitando e/ou contribuindo de forma expressiva para um melhor conhecimento da ecologia microbiana, e os pesquisadores aplicam esse maior conhecimento para entender, desenvolver e aperfeiçoar processos biotecnológicos. Em sistemas de tratamento de esgoto e lixiviados, assim como em solos de cobertura de aterros sanitários, essas técnicas vêm sendo aplicadas no estudo da estrutura e do perfil das comunidades microbianas durante o processo de tratamento desses resíduos. O presente trabalho irá apresentar resumidamente as técnicas mais utilizadas atualmente no estudo de comunidades microbianas nesses ambientes, e relatará algumas novas e promissoras técnicas, com suas potenciais aplicações e limitações

Effect of different salt adaptation strategies on the microbial diversity, activity, and settling of nitrifying sludge in sequencing batch reactors

The effect of salinity on the activity of nitrifying bacteria, floc characteristics, and microbial community structure accessed by fluorescent in situ hybridization and polymerase chain reaction–denaturing gradient gel electrophoresis techniques was investigated. Two sequencing batch reactors (SRB1 and SBR2) treating synthetic wastewater were subjected to increasing salt concentrations. In SBR1, four salt concentrations (5, 10, 15, and 20 g NaCl/L) were tested, while in SBR2, only two salt concentrations (10 and 20 g NaCl/L) were applied in a more shock-wise manner. The two different salt adaptation strategies caused different changes in microbial community structure, but did not change the nitrification performance, suggesting that regardless of the different nitrifying bacterial community present in the reactor, the nitrification process can be maintained stable within the salt range tested. Specific ammonium oxidation rates were more affected when salt increase was performed more rapidly and dropped 50% and 60% at 20 g NaCl/L for SBR1 and SBR2, respectively. A gradual increase in NaCl concentration had a positive effect on the settling properties (i.e., reduction of sludge volume index), although it caused a higher amount of suspended solids in the effluent. Higher organisms (e.g., protozoa, nematodes, and rotifers) as well as filamentous bacteria could not withstand the high salt concentrations

Molecular characterization of Paenibacillus antarcticus IPAC21, a bioemulsifier producer isolated from Antarctic soil

Paenibacillus antarcticus IPAC21, an endospore-forming and bioemulsifier-producing strain, was isolated from King George Island, Antarctica. As psychrotolerant/psychrophilic bacteria can be considered promising sources for novel products such as bioactive compounds and other industrially relevant substances/compounds, the IPAC21 genome was sequenced using Illumina Hi-seq, and a search for genes related to the production of bioemulsifiers and other metabolic pathways was performed. The IPAC21 strain has a genome of 5,505,124 bp and a G + C content of 40.5%. Genes related to the biosynthesis of exopolysaccharides, such as the gene that encodes the extracellular enzyme levansucrase responsible for the synthesis of levan, the 2,3-butanediol pathway, PTS sugar transporters, cold-shock proteins, and chaperones were found in its genome. IPAC21 cell-free supernatants obtained after cell growth in trypticase soy broth at different temperatures were evaluated for bioemulsifier production by the emulsification index (EI) using hexadecane, kerosene and diesel. EI values higher than 50% were obtained using the three oil derivatives when IPAC21 was grown at 28°C. The bioemulsifier produced by P. antarcticus IPAC21 was stable at different NaCl concentrations, low temperatures and pH values, suggesting its potential use in lower and moderate temperature processes in the petroleum industry

Mangrove bacterial diversity and the impact of oil contamination revealed by pyrosequencing:Bacterial proxies for oil pollution

BACKGROUND: Mangroves are transitional coastal ecosystems in tropical and sub-tropical regions and represent biologically important and productive ecosystems. Despite their great ecological and economic importance, mangroves are often situated in areas of high anthropogenic influence, being exposed to pollutants, such as those released by oil spills. METHODOLOGY/PRINCIPAL FINDINGS: A microcosm experiment was conducted, which simulated an oil spill in previously pristine mangrove sediment. The effect of the oil spill on the extant microbial community was studied using direct pyrosequencing. Extensive bacterial diversity was observed in the pristine mangrove sediment, even after oil contamination. The number of different OTUs only detected in contaminated samples was significantly higher than the number of OTUs only detected in non-contaminated samples. The phylum Proteobacteria, in particular the classes Gammaproteobacteria and Deltaproteobacteria, were prevalent before and after the simulated oil spill. On the other hand, the order Chromatiales and the genus Haliea decreased upon exposure to 2 and 5% oil, these are proposed as sensitive indicators of oil contamination. Three other genera, Marinobacterium, Marinobacter and Cycloclasticus increased their prevalence when confronted with oil. These groups are possible targets for the biomonitoring of the impact of oil in mangrove settings. CONCLUSIONS/SIGNIFICANCE: We suggest the use of sequences of the selected genera as proxies for oil pollution, using qPCR assessments. The quantification of these genera in distinct mangrove systems in relation to the local oil levels would permit the evaluation of the level of perturbance of mangroves, being useful in field monitoring. Considering the importance of mangroves to many other environments and the susceptibility of such areas to oil spills this manuscript will be of broad interest

Genomic and phylogenomic insights into the family Streptomycetaceae lead to the proposal of six novel genera

The family Streptomycetaceae is a large and diverse family within the phylum Actinomycetota . The members of the family are known for their ability to produce medically important secondary metabolites, notably antibiotics. In this study, 19 type strains showing low 16S rRNA gene similarity (<97.3 %) to other members of the family Streptomycetaceae were identified and their high genetic diversity was reflected in a phylogenomic analysis using conserved universal proteins. This analysis resulted in the identification of six distinct genus-level clades, with two separated from the genus Streptacidiphilus and four separated from the genus Streptomyces . Compared with members of the genera Streptacidiphilus and Streptomyces , average amino acid identity (AAI) analysis of the novel genera identified gave values within the range of 63.9–71.3 %, as has been previously observed for comparisons of related but distinct bacterial genera. The whole-genome phylogeny was reconstructed using PhyloPhlAn 3.0 based on an optimized subset of conserved universal proteins, the results of AAI and percentage of conserved proteins (POCP) analyses indicated that these phylogenetically distinct taxa may be assigned to six novel genera, namely Actinacidiphila gen. nov., Mangrovactinospora gen. nov., Peterkaempfera gen. nov., Phaeacidiphilus gen. nov., Streptantibioticus gen. nov. and Wenjunlia gen. nov

Search for top squark production in fully hadronic final states in proton-proton collisions at root s=13 TeV

A search for production of the supersymmetric partners of the top quark, top squarks, is presented. The search is based on proton-proton collision events containing multiple jets, no leptons, and large transverse momentum imbalance. The data were collected with the CMS detector at the CERN LHC at a center-of-mass energy of 13 TeV, and correspond to an integrated luminosity of 137 fb(-1). The targeted signal production scenarios are direct and gluino-mediated top squark production, including scenarios in which the top squark and neutralino masses are nearly degenerate. The search utilizes novel algorithms based on deep neural networks that identify hadronically decaying top quarks and W bosons, which are expected in many of the targeted signal models. No statistically significant excess of events is observed relative to the expectation from the standard model, and limits on the top squark production cross section are obtained in the context of simplified supersymmetric models for various production and decay modes. Exclusion limits as high as 1310 GeVare established at the 95% confidence level on the mass of the top squark for direct top squark production models, and as high as 2260 GeV on the mass of the gluino for gluino-mediated top squark production models. These results represent a significant improvement over the results of previous searches for supersymmetry by CMS in the same final state.Peer reviewe