Biobank of fungi from marine and terrestrial Antarctic environments.

Harsh and extreme environments, such as Antarctica, offer unique opportunities to explore new microbial taxa and biomolecules. Given the limited knowledge on microbial diversity, this study aimed to compile, analyze and compare a subset of the biobank of Antarctic fungi maintained at the UNESP's Central of Microbial Resources (CRM-UNESP). A total of 711 isolates (240 yeasts and 471 filamentous fungi) from marine and terrestrial samples collected at King George Island (South Shetland Islands, Antarctica) were used with the primary objective of investigating their presence in both marine and terrestrial environments. Among the yeasts, 13 genera were found, predominantly belonging to the phylum Basidiomycota. Among the filamentous fungi, 34 genera were represented, predominantly from the phylum Ascomycota. The most abundant genera in the marine samples were Metschnikowia, Mrakia, and Pseudogymnoascus, while in the terrestrial samples, they were Pseudogymnoascus, Leucosporidium, and Mortierella. Most of the genera and species of the CRM-UNESP biobank of Antarctic fungi are being reported as an important target for biotechnological applications. This study showed the relevance of the CRM-UNESP biobank, highlighting the importance of applying standard methods for the preservation of the biological material and associated data (BMaD), as recommended in national and international standards.

Antarctic-derived yeasts: taxonomic identification and resistance to adverse conditions.

Antarctic harsh conditions favor the development of microbial adaptations. In this study, a molecular approach was applied to identify/refine the taxonomy of five yeasts isolated from different Antarctic samples, which were tested against ranges of temperature, UV radiations, salinity, and pH. Based on sequencing and phylogenetic analysis, strain CRM 1839 was confirmed as Naganishia sp., and strains CRM 1874, CRM 1565, CRM 2571, and CRM 2576 were identified as Goffeauzyma gilvescens, Goffeauzyma gastrica, Candida atlantica, and Camptobasidium sp., respectively, being this last one possibly a new species. Growth at different temperatures indicates that these yeasts are psychrotolerant, with the exception of Camptobasidium sp., which presents psychrophilic characteristics. G. gastrica recovered from marine sediment showed the best results of resistance to UV radiation, being able to grow even after the exposure to UVB dose of 9144 J/m² and UVC dose of 6102 J/m². C. atlantica isolated from glacier soil showed high cellular growth from 3 to 10% NaCl. The majority of the strains produced higher biomass at pH 7; nevertheless, G. gilvescens showed higher biomass production at pH 9. The studied Antarctic-derived yeasts have adaptations to extreme conditions, which makes them useful for biotechnological applications and studies of extremophiles.

Antarctic fungi applied to textile dye bioremediation.

Antarctica has one of the most hostile conditions on the planet. The environmental characteristics found in this region favor the development of extremophile microorganisms, which are poorly explored biotechnologically. In this context, this study aimed at selectively isolating fungi with potential for the bioremediation of a textile dye. A total of 11 filamentous fungi were isolated from Antarctic samples after incubation in Minimal Mineral medium with the addition of Sulphur Indigo Blue dye. The Antarctic-derived fungi were submitted to textile dye decolorization analysis and biomass production. Isolates LAMAI 2400 and LAMAI 2402 showed more than 90% of decolorization at 15 °C, whereas at 28 °C these isolates showed 81.86 and 98.89%, respectively. In general, the toxicity of the bioassays, evaluated using Cucumis sativus, was higher than in the control. Both isolates, LAMAI 2400 and LAMAI 2402, were identified as Penicillium cf. oxalicum and classified as mesophilic-psychrotolerant. This fungal species has rarely been reported in the Antarctic environments. The results presented herein indicate the potential of the fungi recovered from Antarctic marine sediments for bioremediation of textile dyes at low and moderate temperatures, broadening the perspectives in the field of Antarctic mycology.

Metal resistance mechanisms in Gram-negative bacteria and their potential to remove Hg in the presence of other metals.

Contamination of the environment by heavy metals has been increasing in recent years due to industrial activities. Thus research involving microorganisms capable of surviving in multi-contaminated environments is extremely important. The objectives of the present study were to evaluate the removal of mercury alone and in the presence of cadmium, nickel and lead by four mercury-resistant microorganisms; estimate the removal of Cd, Ni and Pb; understand the mechanisms involved (reduction, siderophores, biofilms, biosorption and bioaccumulation) in the metal resistance of the isolate Pseudomonas sp. B50D; and determine the capacity of Pseudomonas sp. B50D in removing Hg, Cd, Ni and Pb from an industrial effluent. It was shown that the four isolates evaluated were capable of removing from 62% to 95% of mercury from a culture medium with no addition of other metals. The isolate Pseudomonas sp. B50D showed the best performance in the removal of mercury when evaluated concomitantly with other metals. This isolate was capable of removing 75% of Hg in the presence of Cd and 91% in the presence of Ni and Pb. With respect to the other metals it removed 60%, 15% and 85% of Cd, Ni and Pb, respectively. In tests with effluent, the isolate Pseudomonas sp. B50D removed 85% of Hg but did not remove the other metals. This isolate presented reduction, biosorption, biofilm production and siderophore production as its metal resistance mechanisms. Pseudomonas sp. B50D was thus a candidate with potential for application in the bioremediation of effluents with complex metal contaminations.

Methylmercury degradation by Pseudomonas putida V1.

Environmental contamination of mercury (Hg) has caused public health concerns with focuses on the neurotoxic substance methylmercury, due to its bioaccumulation and biomagnification in food chains. The goals of the present study were to examine: (i) the transformation of methylmercury, thimerosal, phenylmercuric acetate and mercuric chloride by cultures of Pseudomonas putida V1, (ii) the presence of the genes merA and merB in P. putida V1, and (iii) the degradation pathways of methylmercury by P. putida V1. Strain V1 cultures readily degraded methylmercury, thimerosal, phenylmercury acetate, and reduced mercuric chloride into gaseous Hg(0). However, the Hg transformation in LB broth by P. putida V1 was influenced by the type of Hg compounds. The merA gene was detected in P. putida V1, on the other hand, the merB gene was not detected. The sequencing of this gene, showed high similarity (100%) to the mercuric reductase gene of other Pseudomonas spp. Furthermore, tests using radioactive (14)C-methylmercury indicated an uncommon release of (14)CO2 concomitant with the production of Hg(0). The results of the present work suggest that P. putida V1 has the potential to remove methylmercury from contaminated sites. More studies are warranted to determine the mechanism of removal of methylmercury by P. putida V1.

Isolation and characterization of bacteria from mercury contaminated sites in Rio Grande do Sul, Brazil, and assessment of methylmercury removal capability of a Pseudomonas putida V1 strain.

Methylmercury (MeHg) is one of the most dangerous heavy metal for living organisms that may be found in environment. Given the crescent industrialization of Brazil and considering that mercury is a residue of several industrial processes, there is an increasing need to encounter and develop remediation approaches of mercury contaminated sites. The aim of this study was to isolate and characterize methylmercury resistant bacteria from soils and sludge sewage from Rio Grande do Sul, Brazil. Sixteen bacteria were isolated from these contaminated sites and some isolates were highly resistant to methylmercury (>8.7 µM). All the isolates were identified by 16S rDNA. Pseudomonas putida V1 was able to volatilize approximately 90 % of methylmercury added to growth media and to resist to copper, lead, nickel, chromate, zinc, cobalt, manganese and barium. In the presence of high concentrations of methylmercury (12 µM), cell growth was limited, but P. putida V1 was still able to remove up to 29 % of this compound from culture medium. This bacterium removed an average of 77 % of methylmercury from culture medium with pH in the range 4.0-6.0. In addition, methylmercury was efficiently removed (>80 %) in temperature of 21-25 °C. Polymerase chain reactions indicated the presence of merA but not merB in P. putida V1. The growth and ability of P. putida V1 to remove methylmercury in a wide range of pH (4.0 and 8.0) and temperature (10-35 °C), its tolerance to other heavy metals and ability to grow in the presence of up to 11.5 µM of methylmercury, suggest this strain as a new potential resource for degrading methylmercury contaminated sites.

A comprehensive study on diesel oil bioremediation under microcosm conditions using a combined microbiological, enzymatic, mass spectrometry, and metabarcoding approach.

This study aims at the application of a marine fungal consortium (Aspergillus sclerotiorum CRM 348 and Cryptococcus laurentii CRM 707) for the bioremediation of diesel oil-contaminated soil under microcosm conditions. The impact of biostimulation (BS) and/or bioaugmentation (BA) treatments on diesel-oil biodegradation, soil quality, and the structure of the microbial community were studied. The use of the fungal consortium together with nutrients (BA/BS) resulted in a TPH (Total Petroleum Hydrocarbon) degradation 42% higher than that obtained by natural attenuation (NA) within 120 days. For the same period, a 72 to 92% removal of short-chain alkanes (C12 to C19) was obtained by BA/BS, while only 3 to 65% removal was achieved by NA. BA/BS also showed high degradation efficiency of long-chain alkanes (C20 to C24) at 120 days, reaching 90 and 92% of degradation of icosane and heneicosane, respectively. In contrast, an increase in the levels of cyclosiloxanes (characterized as bacterial bioemulsifiers and biosurfactants) was observed in the soil treated by the consortium. Conversely, the NA presented a maximum of 37% of degradation of these alkane fractions. The 5-ringed PAH benzo(a)pyrene, was removed significantly better with the BA/BS treatment than with the NA (48 vs. 38 % of biodegradation, respectively). Metabarcoding analysis revealed that BA/BS caused a decrease in the soil microbial diversity with a concomitant increase in the abundance of specific microbial groups, including hydrocarbon-degrading (bacteria and fungi) and also an enhancement in soil microbial activity. Our results highlight the great potential of this consortium for soil treatment after diesel spills, as well as the relevance of the massive sequencing, enzymatic, microbiological and GC-HRMS analyses for a better understanding of diesel bioremediation.

Microbial communities in petroleum-contaminated sites: Structure and metabolisms.

Over recent decades, hydrocarbon concentrations have been augmented in soil and water, mainly derived from accidents or operations that input crude oil and petroleum into the environment. Different techniques for remediation have been proposed and used to mitigate oil contamination. Among the available environmental recovery approaches, bioremediation stands out since these hydrocarbon compounds can be used as growth substrates for microorganisms. In turn, microorganisms can play an important role with significant contributions to the stabilization of impacted areas. In this review, we present the current knowledge about responses from natural microbial communities (using DNA barcoding, multiomics, and functional gene markers) and bioremediation experiments (microcosm and mesocosm) conducted in the presence of petroleum and chemical dispersants in different samples, including soil, sediment, and water. Additionally, we present metabolic mechanisms for aerobic/anaerobic hydrocarbon degradation and alternative pathways, as well as a summary of studies showing functional genes and other mechanisms involved in petroleum biodegradation processes.

Effect of biostimulation and bioaugmentation on hydrocarbon degradation and detoxification of diesel-contaminated soil: a microcosm study.

Soil contamination with diesel oil is quite common during processes of transport and storage. Bioremediation is considered a safe, economical, and environmentally friendly approach for contaminated soil treatment. In this context, studies using hydrocarbon bioremediation have focused on total petroleum hydrocarbon (TPH) analysis to assess process effectiveness, while ecotoxicity has been neglected. Thus, this study aimed to select a microbial consortium capable of detoxifying diesel oil and apply this consortium to the bioremediation of soil contaminated with this environmental pollutant through different bioremediation approaches. Gas chromatography (GC-FID) was used to analyze diesel oil degradation, while ecotoxicological bioassays with the bioindicators Artemia sp., Aliivibrio fischeri (Microtox), and Cucumis sativus were used to assess detoxification. After 90 days of bioremediation, we found that the biostimulation and biostimulation/bioaugmentation approaches showed higher rates of diesel oil degradation in relation to natural attenuation (41.9 and 26.7%, respectively). Phytotoxicity increased in the biostimulation and biostimulation/bioaugmentation treatments during the degradation process, whereas in the Microtox test, the toxicity was the same in these treatments as that in the natural attenuation treatment. In both the phytotoxicity and Microtox tests, bioaugmentation treatment showed lower toxicity. However, compared with natural attenuation, this approach did not show satisfactory hydrocarbon degradation. Based on the microcosm experiments results, we conclude that a broader analysis of the success of bioremediation requires the performance of toxicity bioassays.

Use of digital images to count colonies of biodiesel deteriogenic microorganisms.

This work presents a novel, robust procedure for the semi-automated counting of colony-forming units of Bacillus pumilus (a bacterium) and Meyerozyma guilliermondii (a yeast) both isolated from diesel oil. The counting is performed from digital images of Petri dishes containing the samples by a developed Python code, and the images are acquired from a low-cost scanning apparatus. The counting algorithm is based on the similar morphological characteristics of the bacterium and the yeast colonies. It was compared with classical counting methodology, and the results showed calibration and validation curves with a coefficient of determination (R2) of 0.99 and 0.98, respectively. The developed methodology is a valuable alternative to estimate the microbial contamination of biofuels.

Metal and organic pollutants bioremediation by extremophile microorganisms.

Extremophiles comprise microorganisms that are able to grow and thrive in extreme environments, including in an acidic or alkaline pH, high or low temperatures, high concentrations of pollutants, and salts, among others. These organisms are promising for environmental biotechnology due to their unique physiological and enzymatic characteristics, which allow them to survive in harsh environments. Due to the stability and persistence of these microorganisms under adverse environmental conditions, they can be used for the bioremediation of environments contaminated with extremely recalcitrant pollutants. Here, we provide an overview of extremophiles and the role of "omics" in the field of bioremediation of environmental pollutants, including hydrocarbons, textile dyes and metals.

Metal-resistant rhizobacteria isolates improve Mucuna deeringiana phytoextraction capacity in multi-metal contaminated soils from a gold mining area.

Phytoremediation consists of biological techniques for heavy metal remediation, which include exploring the genetic package of vegetable species to remove heavy metals from the environment. The goals of this study were to investigate heavy metal and bioaugmentation effects on growth and nutrient uptake by Mucuna deeringiana; to determine the metal translocation factor and bioconcentration factor and provide insight for using native bacteria to enhance heavy metal accumulation. The experiment was conducted under greenhouse conditions using a 2 × 4 factorial scheme with highly and slightly contaminated soil samples and inoculating M. deeringiana with three highly lead (Pb+2)-resistant bacteria Kluyvera intermedia (Ki), Klebsiella oxytoca (Ko), and Citrobacter murliniae (Cm) isolated from the rhizosphere of native plants identified as Senecio brasiliensis (Spreng.) Less., Senecio leptolobus DC., and Baccharis trimera (Less) DC., respectively. The increased heavy metal concentrations in soil samples do not decrease the root dry mass of M. deeringiana, concerning the number and dry weight of nodules. The shoot dry mass is reduced by the increasing concentration of heavy metals in soil associated with Kluyvera intermedia and Klebsiella oxytoca bacteria. The number of nodules is affected by heavy metals associated with Citrobacter murliniae bacteria. The bacteria K. intermedia, C. murliniae, and K. oxytoca increase the lead and cadmium available in the soil and enhanced metal uptake by Mucuna deeringiana. The M. deeringiana specie has characteristics that make it hyperaccumulate copper and zinc. The translocation and bioconcentration factors for M. deeringiana characterize it as a promising candidate to phytostabilize multi-metal contaminated soils.

Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A.

This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury.