Extreme environments: a source of biosurfactants for biotechnological applications.

The surfactant industry moves billions of dollars a year and consists of chemically synthesized molecules usually derived from petroleum. Surfactant is a versatile molecule that is widely used in different industrial areas, with an emphasis on the petroleum, biomedical and detergent industries. Recently, interest in environmentally friendly surfactants that are resistant to extreme conditions has increased because of consumers' appeal for sustainable products and industrial processes that often require these characteristics. With this context, the need arises to search for surfactants produced by microorganisms coming from extreme environments and to mine their unique biotechnological potential. The production of biosurfactants is still incipient and presents challenges regarding economic viability due to the high costs of cultivation, production, recovery and purification. Advances can be made by exploring the extreme biosphere and bioinformatics tools. This review focuses on biosurfactants produced by microorganisms from different extreme environments, presenting a complete overview of what information is available in the literature, including the advances, challenges and future perspectives, as well as showing the possible applications of extreme biosurfactants.

The Microbiome of Eucalyptus Roots under Different Management Conditions and Its Potential for Biological Nitrogen Fixation.

Eucalyptus plantations offer a cost-effective and renewable source of raw material. There is substantial interest in improving forestry production, especially through sustainable strategies such as the use of plant growth-promoting bacteria. However, little is known about Eucalyptus microbiology. In this study, the endophytic bacterial community was assessed in Eucalyptus urograndis roots using culture-dependent and culture-independent techniques with plants grown under different conditions. Three phyla accounted for approximately 95% of the community, with Actinobacteria corresponding to approximately 59%. This contrasts with previous studies in which Actinobacteria accounted for only 5 to 10%. Our data also revealed a high diversity of bacteria, with 359 different genera but a high level of dominance. Six genera, Mycobacterium, Bradyrhizobium, Streptomyces, Bacillus, Actinospica, and Burkholderia, accounted for more than 50% of the classified sequences. We observed a significant influence of the treatments on some genera, causing changes in the bacterial community structure. The obtained data also suggest that Eucalyptus may benefit from biological nitrogen fixation, with many abundant genera being closely related to nitrogen-fixing bacteria. Using N-depleted media, we also cultured 95 bacterial isolates, of which 24 tested positive for the nifH gene and were able to maintain growth without any N source in the medium.

Correction to: The Microbiome of Eucalyptus Roots under Different Management Conditions and Its Potential for Biological Nitrogen Fixation.

The original version of this article unfortunately contained mistakes in the first author's name and the running page headers.

Can the Enterococcus faecalis identified in the root canals of primary teeth be a cause of failure of endodontic treatment?

OBJECTIVE: This study investigated the presence of Enterococcus faecalis in primary teeth with primary root canal infections and related to the possible failure of pulpectomy outcome after 36 months. MATERIAL AND METHODS: Root canal samples were obtained from 25 out of 244 patients using the sterile paper cone method. The identification of E. faecalis was done with culture and molecular tests using species-specific 16S rRNA gene-based polymerase chain reaction (PCR). After 36 months, the pulpectomy outcome was evaluated. RESULTS: Enterococcus faecalis was found in five (20%) samples, and dental caries were the cause of primary infection in all of them. Pulpectomy outcome was evaluated only in teeth that completed the entire clinical protocol and were followed up to 36 months (n = 8). From these, 75% (n = 6) were successful and 25% (n = 2) failed. E. faecalis was present in 50% of both successful and failed cases. CONCLUSIONS: Enterococcus faecalis was not related to the failure of endodontic treatment of primary teeth.

Antarctic strict anaerobic microbiota from Deschampsia antarctica vascular plants rhizosphere reveals high ecology and biotechnology relevance.

The Antarctic soil microbial community has a crucial role in the growth and stabilization of higher organisms, such as vascular plants. Analysis of the soil microbiota composition in that extreme environmental condition is crucial to understand the ecological importance and biotechnological potential. We evaluated the efficiency of isolation and abundance of strict anaerobes in the vascular plant Deschampsia antarctica rhizosphere collected in the Antarctic's Admiralty Bay and associated biodiversity to metabolic perspective and enzymatic activity. Using anaerobic cultivation methods, we identified and isolated a range of microbial taxa whose abundance was associated with Plant Growth-Promoting Bacteria (PGPB) and presences were exclusively endemic to the Antarctic continent. Firmicutes was the most abundant phylum (73 %), with the genus Clostridium found as the most isolated taxa. Here, we describe two soil treatments (oxygen gradient and heat shock) and 27 physicochemical culture conditions were able to increase the diversity of anaerobic bacteria isolates. Heat shock treatment allowed to isolate a high percentage of new species (63.63 %), as well as isolation of species with high enzymatic activity (80.77 %), which would have potential industry application. Our findings contribute to the understanding of the role of anaerobic microbes regarding ecology, evolutionary, and biotechnological features essential to the Antarctic ecosystem.

Brazilian Microbiome Project: revealing the unexplored microbial diversity--challenges and prospects.

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world's macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world's catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.

SeqCode in the golden age of prokaryotic systematics.

The SeqCode is a new code of prokaryotic nomenclature that was developed to validate taxon names using genome sequences as the type material. The present article provides an independent view about the SeqCode, highlighting its history, current status, basic features, pros and cons, and use to date. We also discuss important topics to consider for validation of novel prokaryotic taxon names using genomes as the type material. Owing to significant advances in metagenomics and cultivation methods, hundreds of novel prokaryotic species are expected to be discovered in the coming years. This manuscript aims to stimulate and enrich the debate around the use of the SeqCode in the upcoming golden age of prokaryotic taxon discovery and systematics.

Cutting edge tools in the field of soil microbiology.

The study of the whole of the genetic material contained within the microbial populations found in a certain environment is made possible by metagenomics. This technique enables a thorough knowledge of the variety, function, and interactions of microbial communities that are notoriously difficult to research. Due to the limitations of conventional techniques such as culturing and PCR-based methodologies, soil microbiology is a particularly challenging field. Metagenomics has emerged as an effective technique for overcoming these obstacles and shedding light on the dynamic nature of the microbial communities in soil. This review focuses on the principle of metagenomics techniques, their potential applications and limitations in soil microbial diversity analysis. The effectiveness of target-based metagenomics in determining the function of individual genes and microorganisms in soil ecosystems is also highlighted. Targeted metagenomics, including high-throughput sequencing and stable-isotope probing, is essential for studying microbial taxa and genes in complex ecosystems. Shotgun metagenomics may reveal the diversity of soil bacteria, composition, and function impacted by land use and soil management. Sanger, Next Generation Sequencing, Illumina, and Ion Torrent sequencing revolutionise soil microbiome research. Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)'s third and fourth generation sequencing systems revolutionise long-read technology. GeoChip, clone libraries, metagenomics, and metabarcoding help comprehend soil microbial communities. The article indicates that metagenomics may improve environmental management and agriculture despite existing limitations.Metagenomics has revolutionised soil microbiology research by revealing the complete diversity, function, and interactions of microorganisms in soil. Metagenomics is anticipated to continue defining the future of soil microbiology research despite some limitations, such as the difficulty of locating the appropriate sequencing method for specific genes.

Microbiota recovery in a chronosquences of impoverished Cerrado soils with biosolids applications.

Mining activities put the Brazilian savannas, a global biodiversity hotspot, in danger of species and soil carbon losses. Experiments employing biosolids have been applied to rejuvenate this degraded ecosystem, but a lingering question yet to be answered is whether the microbiota that inhabits these impoverished soils can be recovered towards its initial steady state after vegetation recovery. Here, we selected an 18-year-old restoration chronosequence of biosolids-treated, untreated mining and native soils to investigate the soil microbiota recovery based on composition, phylogeny, and diversity, as well as the potential factors responsible for ecosystem recovery. Our results revealed that the soil microbiota holds a considerable recovery potential in the degraded Cerrado biome. Biosolids application not only improved soil health, but also led to 41.7 % recovery of the whole microbial community, featuring significantly higher microbiota diversity and enriched groups (e.g., Firmicutes) that benefit carbon storage compared to untreated mining and native soils. The recovered community showed significant compositional distinctions from the untreated mining or native soils, rather than phylogenetic differences, with physiochemical properties explaining 55 % of the overall community changes. This study advances our understanding of soil microbiota dynamics in response to disturbance and restoration by shedding light on its recovery associated with biosolid application in a degraded biodiverse ecosystem.

Co-inoculation of the endophytes Bacillus thuringiensis CAPE95 and Paenibacillus polymyxa CAPE238 promotes Tropaeolum majus L. growth and enhances its root bacterial diversity.

Tropaeolum majus L. is a versatile edible plant that is widely explored due to its medicinal properties and as a key element in intercropping systems. Its growth could be improved by the use of biofertilizers that can enhance nutrient uptake by the plant or provide tolerance to different abiotic and biotic stresses. In a previous study, 101 endophytes isolated from T. majus roots showed more than three plant growth-promoting (PGP) features in vitro, such as phosphate mineralization/solubilization, production of siderophores, antimicrobial substances and indole-related compounds, and presence of the nifH gene. To provide sustainable alternatives for biofertilization, the genomes of two promising endophytes-CAPE95 and CAPE238-were sequenced to uncover metabolic pathways related to biofertilization. Greenhouse experiments were conducted with 216 seeds and 60 seedlings, half co-inoculated with the endophytes (treatment) and half inoculated with 1X PBS (control), and the impact of the co-inoculation on the plant's bacteriome was accessed through 16S rRNA gene metabarcoding. The strains CAPE95 and CAPE238 were taxonomically assigned as Bacillus thuringiensis and Paenibacillus polymyxa, respectively. Metabolic pathways related to the enhancement of nutrient availability (nitrogen fixation, sulfate-sulfur assimilation), biosynthesis of phytohormones (indole-3-acetic acid precursors) and antimicrobial substances (bacilysin, paenibacillin) were found in their genomes. The in vivo experiments showed that treated seeds exhibited faster germination, with a 20.3% higher germination index than the control on the eleventh day of the experiment. Additionally, treated seedlings showed significantly higher plant height and leaf diameters (p < 0.05). The bacterial community of the treated plants was significantly different from that of the control plants (p < 0.001) and showed a higher richness and diversity of species (Chao and Shannon indexes, p < 0.001). A higher relative abundance of potential synergistic PGP bacteria was also shown in the bacteriome of the treated plants, such as Lysinibacillus and Geobacter. For the first time, co-inoculation of B. thuringiensis and P. polymyxa was shown to have great potential for application as a biofertilizer to T. majus plants. The bacterial consortium used here could also be explored in other plant species in the future.

Investigating Polyextremophilic Bacteria in Al Wahbah Crater, Saudi Arabia: A Terrestrial Model for Life on Saturn's Moon Enceladus.

The study of extremophilic microorganisms has sparked interest in understanding extraterrestrial microbial life. Such organisms are fundamental for investigating life forms on Saturn's icy moons, such as Enceladus, which is characterized by potentially habitable saline and alkaline niches. Our study focused on the salt-alkaline soil of the Al Wahbah crater in Saudi Arabia, where we identified microorganisms that could be used as biological models to understand potential life on Enceladus. The search involved isolating 48 bacterial strains, sequencing the genomes of two thermo-haloalkaliphilic strains, and characterizing them for astrobiological application. A deeper understanding of the genetic composition and functional capabilities of the two novel strains of Halalkalibacterium halodurans provided valuable insights into their survival strategies and the presence of coding genes and pathways related to adaptations to environmental stressors. We also used mass spectrometry with a molecular network approach, highlighting various classes of molecules, such as phospholipids and nonproteinogenic amino acids, as potential biosignatures. These are essential features for understanding life's adaptability under extreme conditions and could be used as targets for biosignatures in upcoming missions exploring Enceladus' orbit. Furthermore, our study reinforces the need to look at new extreme environments on Earth that might contribute to the astrobiology field.

Engineering the mangrove soil microbiome for selection of polyethylene terephthalate-transforming bacterial consortia.

Mangroves are impacted by multiple environmental stressors, including sea level rise, erosion, and plastic pollution. Thus, mangrove soil may be an excellent source of as yet unknown plastic-transforming microorganisms. Here, we assess the impact of polyethylene terephthalate (PET) particles and seawater intrusion on the mangrove soil microbiome and report an enrichment culture experiment to artificially select PET-transforming microbial consortia. The analysis of metagenome-assembled genomes of two bacterial consortia revealed that PET catabolism can be performed by multiple taxa, of which particular species harbored putative novel PET-active hydrolases. A key member of these consortia (Mangrovimarina plasticivorans gen. nov., sp. nov.) was found to contain two genes encoding monohydroxyethyl terephthalate hydrolases. This study provides insights into the development of strategies for harnessing soil microbiomes, thereby advancing our understanding of the ecology and enzymology involved in microbial-mediated PET transformations in marine-associated systems.

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.

Revolutionizing biofuel generation: Unleashing the power of CRISPR-Cas mediated gene editing of extremophiles.

Molecular biology techniques like gene editing have altered the specific genes in micro-organisms to increase their efficiency to produce biofuels. This review paper investigates the outcomes of Clustered regularly interspaced short palindromic repeats (CRISPR) for gene editing in extremophilic micro-organisms to produce biofuel. Commercial production of biofuel from lignocellulosic waste is limited due to various constraints. A potential strategy to enhance the capability of extremophiles to produce biofuel is gene-editing via CRISPR-Cas technology. The efficiency of intracellular enzymes like cellulase, hemicellulose in extremophilic bacteria, fungi and microalgae has been increased by alteration of genes associated with enzymatic activity and thermotolerance. extremophilic microbes like Thermococcus kodakarensis, Thermotoga maritima, Thermus thermophilus, Pyrococcus furiosus and Sulfolobus sp. are explored for biofuel production. The conversion of lignocellulosic biomass into biofuels involves pretreatment, hydrolysis and fermentation. The challenges like off-target effect associated with use of extremophiles for biofuel production is also addressed. The appropriate regulations are required to maximize effectiveness while minimizing off-target cleavage, as well as the total biosafety of this technique. The latest discovery of the CRISPR-Cas system should provide a new channel in the creation of microbial biorefineries through site- specific gene editing that might boost the generation of biofuels from extremophiles. Overall, this review study highlights the potential for genome editing methods to improve the potential of extremophiles to produce biofuel, opening the door to more effective and environmentally friendly biofuel production methods.

A thermophilic chemolithoautotrophic bacterial consortium suggests a mutual relationship between bacteria in extreme oligotrophic environments.

A thermophilic, chemolithoautotrophic, and aerobic microbial consortium (termed carbonitroflex) growing in a nutrient-poor medium and an atmosphere containing N2, O2, CO2, and CO is investigated as a model to expand our understanding of extreme biological systems. Here we show that the consortium is dominated by Carbonactinospora thermoautotrophica (strain StC), followed by Sphaerobacter thermophilus, Chelatococcus spp., and Geobacillus spp. Metagenomic analysis of the consortium reveals a mutual relationship among bacteria, with C. thermoautotrophica StC exhibiting carboxydotrophy and carbon-dioxide storage capacity. C. thermoautotrophica StC, Chelatococcus spp., and S. thermophilus harbor genes encoding CO dehydrogenase and formate oxidase. No pure cultures were obtained under the original growth conditions, indicating that a tightly regulated interactive metabolism might be required for group survival and growth in this extreme oligotrophic system. The breadwinner hypothesis is proposed to explain the metabolic flux model and highlight the vital role of C. thermoautotrophica StC (the sole keystone species and primary carbon producer) in the survival of all consortium members. Our data may contribute to the investigation of complex interactions in extreme environments, exemplifying the interconnections and dependency within microbial communities.

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.

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.

Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste.

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml(-1)). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.

Polyphasic Analysis Reveals Potential Petroleum Hydrocarbon Degradation and Biosurfactant Production by Rare Biosphere Thermophilic Bacteria From Deception Island, an Active Antarctic Volcano.

Extreme temperature gradients in polar volcanoes are capable of selecting different types of extremophiles. Deception Island is a marine stratovolcano located in maritime Antarctica. The volcano has pronounced temperature gradients over very short distances, from as high as 100°C in the fumaroles to subzero next to the glaciers. These characteristics make Deception a promising source of a variety of bioproducts for use in different biotechnological areas. In this study, we isolated thermophilic bacteria from sediments in fumaroles at two geothermal sites on Deception Island with temperatures between 50 and 100°C, to evaluate the potential capacity of these bacteria to degrade petroleum hydrocarbons and produce biosurfactants under thermophilic conditions. We isolated 126 thermophilic bacterial strains and identified them molecularly as members of genera Geobacillus, Anoxybacillus, and Brevibacillus (all in phylum Firmicutes). Seventy-six strains grew in a culture medium supplemented with crude oil as the only carbon source, and 30 of them showed particularly good results for oil degradation. Of 50 strains tested for biosurfactant production, 13 showed good results, with an emulsification index of 50% or higher of a petroleum hydrocarbon source (crude oil and diesel), emulsification stability at 100°C, and positive results in drop-collapse, oil spreading, and hemolytic activity tests. Four of these isolates showed great capability of degrade crude oil: FB2_38 (Geobacillus), FB3_54 (Geobacillus), FB4_88 (Anoxybacillus), and WB1_122 (Geobacillus). Genomic analysis of the oil-degrading and biosurfactant-producer strain FB4_88 identified it as Anoxybacillus flavithermus, with a high genetic and functional diversity potential for biotechnological applications. These initial culturomic and genomic data suggest that thermophilic bacteria from this Antarctic volcano have potential applications in the petroleum industry, for bioremediation in extreme environments and for microbial enhanced oil recovery (MEOR) in reservoirs. In addition, recovery of small-subunit rRNA from metagenomes of Deception Island showed that Firmicutes is not among the dominant phyla, indicating that these low-abundance microorganisms may be important for hydrocarbon degradation and biosurfactant production in the Deception Island volcanic sediments.