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.

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.

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.

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.

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.

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.

Pseudomonas fluorescens Complex and Its Intrinsic, Adaptive, and Acquired Antimicrobial Resistance Mechanisms in Pristine and Human-Impacted Sites.

Pseudomonas spp. are ubiquitous microorganisms that exhibit intrinsic and acquired resistance to many antimicrobial agents. Pseudomonas aeruginosa is the most studied species of this genus due to its clinical importance. In contrast, the Pseudomonas fluorescens complex consists of environmental and, in some cases, pathogenic opportunistic microorganisms. The records of antimicrobial-resistant P. fluorescens are quite scattered, which hinders the recognition of patterns. This review compiles published data on antimicrobial resistance in species belonging to the P. fluorescens complex, which were identified through phylogenomic analyses. Additionally, we explored the occurrence of clinically relevant antimicrobial resistance genes in the genomes of the respective species available in the NCBI database. Isolates were organized into two categories: strains isolated from pristine sites and strains isolated from human-impacted or metal-polluted sites. Our review revealed that many reported resistant phenotypes in this complex might be related to intrinsic features, whereas some of them might be ascribed to adaptive mechanisms such as colistin resistance. Moreover, a few studies reported antimicrobial resistance genes (ARGs), mainly ß-lactamases. In-silico analysis corroborated the low occurrence of transferable resistance mechanisms in this Pseudomonas complex. Both phenotypic and genotypic assays are necessary to gain insights into the evolutionary aspects of antimicrobial resistance in the P. fluorescens complex and the possible role of these ubiquitous species as reservoirs of clinically important and transmissible ARGs.

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.

Brazilian Semi-Arid Mangroves-Associated Microbiome as Pools of Richness and Complexity in a Changing World.

Mangrove microbiomes play an essential role in the fate of mangroves in our changing planet, but the factors regulating the biogeographical distribution of mangrove microbial communities remain essentially vague. This paper contributes to our understanding of mangrove microbiomes distributed along three biogeographical provinces and ecoregions, covering the exuberant mangroves of Amazonia ecoregion (North Brazil Shelf) as well as mangroves located in the southern limit of distribution (Southeastern ecoregion, Warm Temperate Southwestern Atlantic) and mangroves localized on the drier semi-arid coast (Northeastern ecoregion, Tropical Southwestern Atlantic), two important ecotones where poleward and landward shifts, respectively, are expected to occur related to climate change. This study compared the microbiomes associated with the conspicuous red mangrove (Rhizophora mangle) root soils encompassing soil properties, latitudinal factors, and amplicon sequence variants of 105 samples. We demonstrated that, although the northern and southern sites are over 4,000 km apart, and despite R. mangle genetic divergences between north and south populations, their microbiomes resemble each other more than the northern and northeastern neighbors. In addition, the northeastern semi-arid microbiomes were more diverse and displayed a higher level of complexity than the northern and southern ones. This finding may reflect the endurance of the northeast microbial communities tailored to deal with the stressful conditions of semi-aridity and may play a role in the resistance and growing landward expansion observed in such mangroves. Minimum temperature, precipitation, organic carbon, and potential evapotranspiration were the main microbiota variation drivers and should be considered in mangrove conservation and recovery strategies in the Anthropocene. In the face of changes in climate, land cover, biodiversity, and chemical composition, the richness and complexity harbored by semi-arid mangrove microbiomes may hold the key to mangrove adaptability in our changing planet.

Proposal of Carbonactinosporaceae fam. nov. within the class Actinomycetia. Reclassification of Streptomyces thermoautotrophicus as Carbonactinospora thermoautotrophica gen. nov., comb. nov.

Streptomyces thermoautotrophicus UBT1T has been suggested to merit generic status due to its phylogenetic placement and distinctive phenotypes among Actinomycetia. To evaluate whether 'S. thermoautotrophicus' represents a higher taxonomic rank, 'S. thermoautotrophicus' strains UBT1T and H1 were compared to Actinomycetia using 16S rRNA gene sequences and comparative genome analyses. The UBT1T and H1 genomes each contain at least two different 16S rRNA sequences, which are closely related to those of Acidothermus cellulolyticus (order Acidothermales). In multigene-based phylogenomic trees, UBT1T and H1 typically formed a sister group to the Streptosporangiales-Acidothermales clade. The Average Amino Acid Identity, Percentage of Conserved Proteins, and whole-genome Average Nucleotide Identity (Alignment Fraction) values were ≤58.5%, ≤48%, ≤75.5% (0.3) between 'S. thermoautotrophicus' and Streptosporangiales members, all below the respective thresholds for delineating genera. The values for genomics comparisons between strains UBT1T and H1 with Acidothermales, as well as members of the genus Streptomyces, were even lower. A review of the 'S. thermoautotrophicus' proteomic profiles and KEGG orthology demonstrated that UBT1T and H1 present pronounced differences, both tested and predicted, in phenotypic and chemotaxonomic characteristics compared to its sister clades and Streptomyces. The distinct phylogenetic position and the combination of genotypic and phenotypic characteristics justify the proposal of Carbonactinospora gen. nov., with the type species Carbonactinospora thermoautotrophica comb. nov. (type strain UBT1T, = DSM 100163T = KCTC 49540T) belonging to Carbonactinosporaceae fam. nov. within Actinomycetia.

Skin and stinger bacterial communities in two critically endangered rays from the South Atlantic in natural and aquarium settings.

Bacterial communities of two critically endangered rays from the South Atlantic, the butterfly ray (Gymnura altavela) and the groovebelly ray (Dasyatis hypostigma), were described using 16S rRNA gene metabarcoding. The study characterized the bacterial communities associated with (i) G. altavela in natural (in situ) and aquarium (ex situ) settings, (ii) skin and stinger of G. altavela, and D. hypostigma in aquaria, and (iii) newborns and adults of D. hypostigma. The results revealed potentially antibiotic-producing bacterial groups on the skin of rays from the natural environment, and some taxa with the potential to benefit ray health, mainly in rays from the natural environment, as well as possible pathogens to other animals, including fish and humans. Differences were observed between the G. altavela and D. hypostigma bacteria composition, as well as between the skin and stinger bacterial composition. The bacterial community associated with D. hypostigma changed with the age of the ray. The aquarium environment severely impacted the G. altavela bacteria composition, which changed from a complex bacterial community to one dominated almost exclusively by two taxa, Oceanimonas sp. and Sediminibacterium sp. on the skin and stinger, respectively.

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.

Tracking Mangrove Oil Bioremediation Approaches and Bacterial Diversity at Different Depths in an in situ Mesocosms System.

In this study, oil spills were simulated in field-based mangrove mesocosms to compare the efficiency of bioremediation strategies and to characterize the presence of the alkB, ndo, assA, and bssA genes and the ecological structures of microbial communities in mangrove sediments at two different depths, (D1) 1-10 cm and (D2) 25-35 cm. The results indicated that the hydrocarbon degradation efficiency was higher in superficial sediment layers, although no differences in the hydrocarbon degradation rates or in the abundances of the alkB and ndo genes were detected among the tested bioremediation strategies at this depth. Samples from the deeper layer exhibited higher abundances of the analyzed genes, except for assA and bssA, which were not detected in our samples. For all of the treatments and depths, the most abundant phyla were Proteobacteria, Firmicutes and Bacteroidetes, with Gammaproteobacteria, Flavobacteriales and Clostridiales being the most common classes. The indicator species analysis (ISA) results showed strong distinctions among microbial taxa in response to different treatments and in the two collection depths. Our results indicated a high efficiency of the monitored natural attenuation (MNA) for oil consumption in the tested mangrove sediments, revealing the potential of this strategy for environmental decontamination and suggesting that environmental and ecological factors may select for specific bacterial populations in distinct niches.

Draft Genome Sequence of Geobacillus sp. Strain LEMMJ02, a Thermophile Isolated from Deception Island, an Active Volcano in Antarctica.

The thermophilic Geobacillus sp. strain LEMMJ02 was isolated from Fumarole Bay sediment on Deception Island, an active Antarctic volcano. Here, we report the draft genome of LEMMJ02, which consists of 3,160,938 bp with 52.8% GC content and 3,523 protein-coding genes.

Draft Genome Sequence of Brevibacillus sp. Strain LEMMJ03, Isolated from an Antarctic Volcano.

Here, we announce the draft genome sequence of Brevibacillus sp. strain LEMMJ03, isolated from Whalers Bay sediment (Deception Island, Antarctica). In total, 4,500 coding sequences (CDS), among those 102 coding for tRNAs and 5 for noncoding RNAs (ncRNAs), were predicted from the 4.64-Mb genome. Predicted functions were for bacteriocin and degradation of aromatic compounds.

Carotenoids from UV-resistant Antarctic Microbacterium sp. LEMMJ01.

The Microbacterium sp. LEMMJ01 isolated from Antarctic soil does not belong to any of the nearest species identified in the RDP database. Under UV radiation (A, B and C wavebands) the survival fractions of Microbacterium sp. cells were much higher compared with wild-type E. coli K12A15. Especially remarkable for an Antarctic bacterium, an expressive resistance against high UV-B doses was observed. The increased survival of DNA repair-proficient E. coli grown overnight added of 0.1 mg/ml or 1 mg/ml of the whole pigment extract produced by Microbacterium sp. revealed that part of the resistance of Microbacterium sp. against UV-B radiation seems to be connected with photoprotection by its pigments. Scanning electron microscopy revealed that UV-A and UV-B ensued membrane alterations only in E. coli. The APCI-MS fingerprints revealed the diagnostic ions for neurosporene (m/z 580, 566, 522, 538, and 524) synergism for the first time in this bacterium by HPLC-MS/MS analysis. Carotenoids also were devoid of phototoxicity and cytotoxicity effects in mouse cells and in human keratinocytes and fibroblasts.

Fungal communities in oil contaminated mangrove sediments - Who is in the mud?

Mangroves are ecosystems located in tropical and subtropical regions of the world and are vital for coastal protection. Their unique characteristics make them hotspots for carbon cycling and biological diversity. Studies on isolated filamentous fungi and environmental and anthropogenic factors that influence sediments offer new understandings on how to preserve mangroves. Here we report on the filamentous fungi isolated from four mangroves. We correlated fungal community composition with sediment texture, polycyclic aromatic hydrocarbons concentration (oil pollution), pH, salinity, organic matter, total and thermotolerant coliforms (sewage pollution). In total we identified 34 genera and 97 species. The most polluted sites had highest species richness whereas the best preserved site showed the lowest species richness. Oil spill and sewage pollution were identified as the drivers of fungal community composition in the most polluted sites. We found very distinct fungal communities with no >5 species shared between any two mangrove sites.

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.

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.