Phosphate solubilization by Antarctic yeasts isolated from lichens.

Antarctica has a great diversity of microorganisms with biotechnological potential but is not very well Known about yeasts with phosphate solubilization activity. Thus, the aim of this study was to evaluate the ability of yeasts from Antarctica lichens to solubilize phosphate in vitro. In the screening, 147 yeasts were tested and 43 (29%) showed P solubilization in solid NBRIP medium at 15.0 °C, with a higher prevalence of positive genera Vishniacozyma, followed by Cystobasidium. Most of the positive yeasts were isolated from Usnea auratiacoatra, followed by Polycauliona regalis and Lecania brialmontii. Two strains with better activity after screening were selected for the solubilization in the liquid medium, Vishniacozyma victoriae 2.L15 and A.L6 (unidentified). Vishniacozyma victoriae 2.L15 exhibiting activities at 25.0 °C (29.91 mg/L of phosphate and pH 6.85) and at 30.0 °C (619.04 mg/L of phosphate and pH 3.73) and A.L6 strain at 25.0 °C (25.05 mg/L of phosphate and pH 6.69) and at 30.0 °C (31.25 mg/L of phosphate and pH 6.47). Of eight organic acids tested by HPLC, tartaric and acetic acids were detected during phosphate solubilization, with greater release in the period of 144 (2.13 mg/L) and 72 (13.72 mg/L) hours, respectively. Future studies to elucidate the presence of functional genes for P metabolism in lichens, as well as studies in the field of proteomics for the discovery of yeast proteins related to P solubilization are needed. Thus, the high prevalence of lichen-associated yeast communities probably contributed to the high frequency of phosphate-solubilizing isolates in this study.

Pharmaceutical biotechnological potential of filamentous fungi isolated from textile industry.

The need for more effective drugs for the treatment of infectious diseases as well as for general applications including wound healing and burn surgery, has guided efforts for the discovery of new compounds of medical interest. Microorganisms found in textile industrial waste have the ability to produce a variety of enzymes and/or secondary metabolites including molecules of pharmaceutical interest. The present work investigated the biotechnological potential of filamentous fungi isolated from textile industry wastewater for the production of collagenase and antimicrobial metabolites. From 28 isolates assayed, Sarocladium sp. ITF33 showed specific collagenolytic activity with values of 7.62 and 9.04 U mg-1 for the intracellular and extracellular fractions, respectively. The isolate Penicillium sp. ITF28 showed the best antimicrobial activity, reaching MIC ranging from 1.0 to 0.0625 mg mL-1 against five pathogenic bacteria. Molecular analyzes suggest that the isolate Sarocladium sp. ITF 33 can be considered a species not yet described. The results of the present work encourage studies of characterization and purification of the enzymes and secondary metabolites produced by the isolates found aiming future applications in the medical and pharmaceutical fields.

Metagenomic Insights Into the Mechanisms for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Oil Supply Chain.

Petroleum is a very complex and diverse organic mixture. Its composition depends on reservoir location and in situ conditions and changes once crude oil is spilled into the environment, making the characteristics associated with every spill unique. Polycyclic aromatic hydrocarbons (PAHs) are common components of the crude oil and constitute a group of persistent organic pollutants. Due to their highly hydrophobic, and their low solubility tend to accumulate in soil and sediment. The process by which oil is sourced and made available for use is referred to as the oil supply chain and involves three parts: (1) upstream, (2) midstream and (3) downstream activities. As consequence from oil supply chain activities, crude oils are subjected to biodeterioration, acidification and souring, and oil spills are frequently reported affecting not only the environment, but also the economy and human resources. Different bioremediation techniques based on microbial metabolism, such as natural attenuation, bioaugmentation, biostimulation are promising approaches to minimize the environmental impact of oil spills. The rate and efficiency of this process depend on multiple factors, like pH, oxygen content, temperature, availability and concentration of the pollutants and diversity and structure of the microbial community present in the affected (contaminated) area. Emerging approaches, such as (meta-)taxonomics and (meta-)genomics bring new insights into the molecular mechanisms of PAH microbial degradation at both single species and community levels in oil reservoirs and groundwater/seawater spills. We have scrutinized the microbiological aspects of biodegradation of PAHs naturally occurring in oil upstream activities (exploration and production), and crude oil and/or by-products spills in midstream (transport and storage) and downstream (refining and distribution) activities. This work addresses PAH biodegradation in different stages of oil supply chain affecting diverse environments (groundwater, seawater, oil reservoir) focusing on genes and pathways as well as key players involved in this process. In depth understanding of the biodegradation process will provide/improve knowledge for optimizing and monitoring bioremediation in oil spills cases and/or to impair the degradation in reservoirs avoiding deterioration of crude oil quality.

Actinobacteria from Antarctica as a source for anticancer discovery.

Although many advances have been achieved to treat aggressive tumours, cancer remains a leading cause of death and a public health problem worldwide. Among the main approaches for the discovery of new bioactive agents, the prospect of microbial secondary metabolites represents an effective source for the development of drug leads. In this study, we investigated the actinobacterial diversity associated with an endemic Antarctic species, Deschampsia antarctica, by integrated culture-dependent and culture-independent methods and acknowledged this niche as a reservoir of bioactive strains for the production of antitumour compounds. The 16S rRNA-based analysis showed the predominance of the Actinomycetales order, a well-known group of bioactive metabolite producers belonging to the Actinobacteria phylum. Cultivation techniques were applied, and 72 psychrotolerant Actinobacteria strains belonging to the genera Actinoplanes, Arthrobacter, Kribbella, Mycobacterium, Nocardia, Pilimelia, Pseudarthrobacter, Rhodococcus, Streptacidiphilus, Streptomyces and Tsukamurella were identified. The secondary metabolites were screened, and 17 isolates were identified as promising antitumour compound producers. However, the bio-guided assay showed a pronounced antiproliferative activity for the crude extracts of Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653. The TGI and LC50 values revealed the potential of these natural products to control the proliferation of breast (MCF-7), glioblastoma (U251), lung/non-small (NCI-H460) and kidney (786-0) human cancer cell lines. Cinerubin B and actinomycin V were the predominant compounds identified in Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653, respectively. Our results suggest that the rhizosphere of D. antarctica represents a prominent reservoir of bioactive actinobacteria strains and reveals it as an important environment for potential antitumour agents.

In depth metagenomic analysis in contrasting oil wells reveals syntrophic bacterial and archaeal associations for oil biodegradation in petroleum reservoirs.

Microbial biodegradation of hydrocarbons in petroleum reservoirs has major consequences in the petroleum value and quality. The identification of microorganisms capable of in-situ degradation of hydrocarbons under the reservoir conditions is crucial to understand microbial roles in hydrocarbon transformation and the impact of oil exploration and production on energy resources. The aim of this study was to profile the metagenome of microbial communities in crude oils and associated formation water from two high temperature and relatively saline oil-production wells, where one has been subjected to water flooding (BA-2) and the other one is considered pristine (BA-1). The microbiome was studied in the fluids using shotgun metagenome sequencing. Distinct microbial compositions were revealed when comparing pristine and water flooded oil wells in contrast to the similar community structures observed between the aqueous and oil fluids from the same well (BA-2). The equal proportion of archaea and bacteria together with the greater anaerobic hydrocarbon degradation potential in the BA-1 pristine but degraded reservoir contrasted with the predominance of bacteria over archaea, aerobic pathways and lower frequency of anaerobic degradation genes in the BA-2 water flooded undegraded well. Our results suggest that Syntrophus, Syntrophomonas, candidatus Atribacteria and Synergistia, in association with mainly acetoclastic methanogenic archaea of the genus Methanothrix, were collectively responsible for the oil biodegradation observed in the pristine petroleum well BA-1. Conversely, the microbial composition of the water flooded oil well BA-2 was mainly dominated by the fast-growing and putatively aerobic opportunists Marinobacter and Marinobacterium. This presumable allochthonous community introduced a greater metabolic versatility, although oil biodegradation has not been detected hitherto perhaps due to in-reservoir unfavorable physicochemical conditions. These findings provide a better understanding of the petroleum reservoir microbiomes and their potential roles in biogeochemical processes occurring in environments with different geological and oil recovery histories.

Hydrocarbon-associated substrates reveal promising fungi for poly (ethylene terephthalate) (PET) depolymerization.

Recalcitrant characteristics and insolubility in water make the disposal of synthetic polymers a great environmental problem to be faced by modern society. Strategies towards the recycling of post-consumer polymers, like poly (ethylene terephthalate, PET) degradation/depolymerization have been studied but still need improvement. To contribute with this purpose, 100 fungal strains from hydrocarbon-associated environments were screened for lipase and esterase activities by plate assays and high-throughput screening (HTS), using short- and long-chain fluorogenic probes. Nine isolates were selected for their outstanding hydrolytic activity, comprising the genera Microsphaeropsis, Mucor, Trichoderma, Westerdykella, and Pycnidiophora. Two strains of Microsphaeropsis arundinis were able to convert 2-3% of PET nanoparticle into terephthalic acid, and when cultured with two kinds of commercial PET bottle fragments, they also promoted weight loss, surface and chemical changes, increased lipase and esterase activities, and led to PET depolymerization with release of terephthalic acid at concentrations above 20.0 ppm and other oligomers over 0.6 ppm. The results corroborate that hydrocarbon-associated areas are important source of microorganisms for application in environmental technologies, and the sources investigated revealed important strains with potential for PET depolymerization.

A Novel Multifunctional ß-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove.

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a ß-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a ß-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-ß-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-ß-d-galactopyranoside, and 4-Nitrophenyl ß-d-glucopyranoside, presenting ß-N-acetylglucosaminidase, ß-glucosidase, and ß-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel ß-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

Functional metagenomics of oil-impacted mangrove sediments reveals high abundance of hydrolases of biotechnological interest.

Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms. For this, an in-depth survey of the taxonomic and functional microbial diversity recovered in a fosmid library (Library_Oil Mgv) constructed from oil-impacted Brazilian mangrove sediment was carried out. Fosmid DNA of the whole library was extracted and submitted to Illumina HiSeq sequencing. The resulting Library Oil_Mgv dataset was further compared with those obtained by direct sequencing of environmental DNA from Brazilian mangroves (from distinct regions and affected by distinct sources of contamination), focusing on hydrolases with potential use in biotechnological processes. The most abundant hydrolases found were proteases, esterases and amylases, with similar occurrence profile in all datasets. The main microbial groups harboring such hydrolase-encoding genes were distinct in each mangrove, and in the fosmid library these enzymes were mainly assigned to Chloroflexaceae (for amylases), Planctomycetaceae (for esterases) and Bradyrhizobiaceae (for proteases). Assembly and analysis of Library_Oil Mgv reads revealed three potentially novel enzymes, one epoxide hydrolase, one xylanase and one amylase, to be further investigated via heterologous expression assays.

Compositional profile of α / ß-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

The occurrence of genes encoding biotechnologically relevant α/ß-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/ß-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes.

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs.

This study aimed at evaluating potential differences among the bacterial communities from formation water and oil samples originated from biodegraded and non-biodegraded Brazilian petroleum reservoirs by using a PCR-DGGE based approach. Environmental DNA was isolated and used in PCR reactions with bacterial primers, followed by separation of 16S rDNA fragments in the DGGE. PCR products were also cloned and sequenced, aiming at the taxonomic affiliation of the community members. The fingerprints obtained allowed the direct comparison among the bacterial communities from oil samples presenting distinct degrees of biodegradation, as well as between the communities of formation water and oil sample from the non-biodegraded reservoir. Very similar DGGE band profiles were observed for all samples, and the diversity of the predominant bacterial phylotypes was shown to be low. Cloning and sequencing results revealed major differences between formation water and oil samples from the non-biodegraded reservoir. Bacillus sp. and Halanaerobium sp. were shown to be the predominant components of the bacterial community from the formation water sample, whereas the oil sample also included Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. and Acidithiobacillus ferrooxidans. The PCR-DGGE technique, combined with cloning and sequencing of PCR products, revealed the presence of taxonomic groups not found previously in these samples when using cultivation-based methods and 16S rRNA gene library assembly, confirming the need of a polyphasic study in order to improve the knowledge of the extent of microbial diversity in such extreme environments.