Petroleum Hydrocarbon Bioremediation Using Native Fungal Isolates and Consortia.

Crude oil spills as a result of natural disasters or extraction and transportation operations are common nowadays. Oil spills have adverse effects on both aquatic and terrestrial ecosystems and pose a threat to human health. This study have been concerned with studying the capability of six fungal species (Curvularia brachyspora, Penicillium chrysogenum, Scopulariopsis brevicaulis, Cladosporium sphaerospermum, Alternaria alternata, and Stemphylium botryosum) and three fungal consortia (FC), FC1 (P. chrysogenum and C. brachyspora), FC2 (S. brevicaulis and S. botryosum), and FC3 (S. brevicaulis, S. botryosum, and C. sphaerospermum), to remediate petroleum hydrocarbons (PHs). Qualitative and quantitative changes in polyaromatic hydrocarbons (PAHs) and saturated hydrocarbons (SH) mixtures and the patterns of PHs degradation have been examined using HPLC and GC. Studying the GC chromatogram of C. sphaerospermum revealed severe degradation of SHs exhibited by this species, and the normal-paraffin and isoparaffin degradation percentage have been valued 97.19% and 98.88%, respectively. A. alternata has shown the highest significant (at P ˂ 0.05) PAH degradation percent reaching 72.07%; followed by P. chrysogenum, 59.51%. HPLC data have revealed that high-molecular-weight PAH percent/total PAHs decreased significantly from 98.94% in control samples to 68.78% in samples treated with A. alternata. FC1 and FC2 consortia have exhibited the highest significant PH deterioration abilities than did the individual isolates, indicating that these fungal consortia exhibited positive synergistic effects. The study supports the critical idea of the potential PAH and SH biodegradation as a more ecologically acceptable alternative to their chemical degradation.

Linking Pélardon artisanal goat cheese microbial communities to aroma compounds during cheese-making and ripening.

Pélardon is an artisanal French raw goat's milk cheese, produced using natural whey as a backslop. The aim of this study was to identify key microbial players involved in the acidification and aroma production of this Protected Designation of Origin cheese. Microbial diversity of samples, collected from the raw milk to 3-month cheese ripening, was determined by culture-dependent (MALDI-TOF analysis of 2877 isolates) and -independent (ITS2 and 16S metabarcoding) approaches and linked to changes in biochemical profiles (volatile compounds and acids). In parallel, potential dominant autochthonous microorganism reservoirs were also investigated by sampling the cheese-factory environment. Complex and increasing microbial diversity was observed by both approaches during ripening although major discrepancies were observed regarding Lactococcus lactis and Lacticaseibacillus paracasei fate. By correlating microbial shifts to biochemical changes, Lactococcus lactis was identified as the main acidifying bacterium, while L. mesenteroides and Geotrichum candidum were prevalent and associated with amino acids catabolism after the acidification step. The three species were dominant in the whey (backslop). In contrast, L. paracasei, Enterococcus faecalis, Penicillium commune and Scopulariopsis brevicaulis, which dominated during ripening, likely originated from the cheese-making environment. All these four species were positively correlated to major volatile compounds responsible for the goaty and earthy Pélardon cheese aroma. Overall, this work highlighted the power of MALDI-TOF and molecular techniques combined with volatilome analyses to dynamically follow and identify microbial communities during cheese-making and successively identify the key-players involved in aroma production and contributing to the typicity of Pélardon cheese.

An application of advanced hair-save processes in leather industry as the reason of formation of keratinous waste: few peculiarities of its utilisation.

The application of more environmentally friendly hide and skin unhairing technologies in leather processing results in a significant increase in keratin waste. There are currently two most promising hair-saving unhairing methods: enzymatic and hair immunisation. The complete use of hair-saving unhairing methods in the leather industry will lead to the formation of approximately 143 thousand tons of hair/wool waste annually, which will require disposal. The disposal of keratin wastes from the leather industry has not been adequately studied, bearing in mind the possible amount of such wastes that will be produced in the future. Unfortunately, existing studies pay little attention to the method of unhairing, even though the unhairing method has a vast influence on the properties of keratin in the obtained hair/wool wastes. Accordingly, the present research is an attempt to establish how the differently obtained keratin wastes behave following disposal. The obtained results have shown that waste wool is characterised by different behaviour during burial in soil, and the behaviour depends on the method of unhairing. This proposition is valid for waste wool bioresistance as well. It was concluded that the deterioration of any sort of keratinous waste from the leather industry should be investigated thoroughly before disposal by burial in landfills.

Scopuquinolone B, a new monoterpenoid dihydroquinolin-2(1H)-one isolated from the coral-derived Scopulariopsis sp. fungus.

Further chemical investigation of the secondary metabolites of the fungus Scopulariopsis sp. led to the discovery of a new alkaloid, scopuquinolone B (1). The structure of compound 1 was elucidated by extensive NMR spectroscopic data, CD spectrum and analysis of its Dess-Martin oxidation derivative. Compound 1 was evaluated for antilarval settlement activity of barnacle Balanus amphitrite and showed promising antifouling activity with an EC50 value of 0.103 µM and a high therapeutic ratio of 222.

Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species.

Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes. IMPORTANCE: Decades of culture-based studies and more recent metagenomic studies have demonstrated that bacterial species in agriculture, medicine, industry, and nature are unevenly distributed across time and space. The ecological processes and molecular mechanisms that shape these distributions are not well understood because it is challenging to connect in situ patterns of diversity with mechanistic in vitro studies in the laboratory. Using tractable cheese rind biofilms and a focus on coagulase-negative staphylococcus (CNS) species, we demonstrate that fungi can mediate the ecological distributions of closely related bacterial species. One of the Staphylococcus species studied, S. saprophyticus, is a common cause of urinary tract infections. By identifying processes that control the abundance of undesirable CNS species, cheese producers will have more precise control on the safety and quality of their products. More generally, Staphylococcus species frequently co-occur with fungi in mammalian microbiomes, and similar bacterium-fungus interactions may structure bacterial diversity in these systems.

Depletion of pentachlorophenol in soil microcosms with Byssochlamys nivea and Scopulariopsis brumptii as detoxification agents.

Pentachlorophenol (PCP) is a toxic compound which is widely used as a wood preservative product and general biocide. It is persistent in the environment and has been classified as a persistent organic pollutant to be reclaimed in many countries. Fungal bioremediation is an emerging approach to rehabilitating areas fouled by recalcitrant xenobiotics. In the present study, we isolated two fungal strains from an artificially PCP-contaminated soil during a long-term bioremediation study and evaluated their potential as bioremediation agents in depletion and detoxification of PCP in soil microcosms. The two fungal strains were identified as: Byssochlamys nivea (Westling, 1909) and Scopulariopsis brumptii (Salvanet-Duval, 1935). PCP removal and toxicity were examined during 28 days of incubation. Bioaugmented microcosms revealed a 60% and 62% PCP removal by B. nivea and S. brumptii, respectively. Co-inoculation of B. nivea and S. brumptii showed a synergetic effect on PCP removal resulting in 95% and 80% PCP decrease when initial concentrations were 12.5 and 25 mg kg-1, respectively. Detoxification in bioaugmented soil and the efficient role of fungi in the rehabilitation of PCP contaminated soil were experimentally proven by toxicity assays. A decrease in inhibition of bioluminescence of Escherichia coli HB101 pUCD607 and an increase of germination index of mustard (Brassica alba) seeds were observed in the decontaminated soils.

Phylogenetic Relationship and Secondary Metabolite Production of Marine Fungi Producing the Cyclodepsipeptides Scopularide A and B.

Strains originally affiliated to the genera Scopulariopsis and Microascus were compared regarding the scopularide production in order to investigate their ability to produce the cyclodepsipeptides and select the best suited candidate for subsequent optimisation processes. Phylogenetic calculations using available sequences of the genera Scopulariopsis and Microascus revealed that most of the sequences clustered within two closely related groups, comprising mainly Scopulariopsis/Microascus brevicaulis and Microascus sp., respectively. Interestingly, high yields of scopularide A were exhibited by three strains belonging to S./M. brevicaulis, while lower titres were observed for two strains of Microascus sp. Close phylogenetic distances within and between the two groups supported the proposed combination of both genera into one holomorph group. Short phylogenetic distances did not allow a clear affiliation at the species level on the basis of ribosomal DNA sequences, especially for Microascus sp. strains. Additionally, several sequences originating from strains assigned to Scopulariopsis exhibited a polyphyletic nature. The production pattern is in accordance with the phylogenetic position of the strains and significant production of scopularide B could only be observed for the S./M. brevicaulis strain LF580. Thus, the phylogenetic position marks the biotechnologically interesting strains and matters in optimisation strategies. In conclusion, the ability of all five strains to produce at least one of the scopularides suggests a distribution of the responsible gene cluster within the holomorph group. Setting the focus on the production of the cyclodepsipeptides, strain LF580 represents the best candidate for further strain and process optimisation.

De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster.

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis.

Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

Development and validation of a fast and optimized screening method for enhanced production of secondary metabolites using the marine Scopulariopsis brevicaulis strain LF580 producing anti-cancer active scopularide A and B.

Natural compounds from marine fungi are an excellent source for the discovery and development of new drug leads. The distinct activity profiles of the two cyclodepsipeptides scopularide A and B against cancer cell lines set their marine producer strain Scopulariopsis brevicaulis LF580 into the focus of the EU project MARINE FUNGI. One of the main goals was the development of a sustainable biotechnological production process for these compounds. The secondary metabolite production of strain LF580 was optimized by random mutagenesis employing UV radiation. For a fast and reliable detection of the intracellular secondary metabolite production level, a miniaturized bioactivity-independent screening method was developed, as the random mutagenesis yielded a large number of mutants to be analysed quantitatively and none of the existing hyphenated bioassay-dependent screening systems could be applied. The method includes decreased cultivation volume, a fast extraction procedure as well as an optimized LC-MS analysis. We show that deviation could be specifically reduced at each step of the process: The measuring deviation during the analysis could be minimized to 5% and technical deviation occurring in the downstream part to 10-15%. Biological variation during the cultivation process still has the major influence on the overall variation. However, the approach led to a 10-fold reduction of time and similar effects on costs and effort compared to standard reference screening methods. The method was applied to screen the UV-mutants library of Scopulariopsis brevicaulis LF580. For validation purposes, the occurring variations in the miniaturized scale were compared to those in the classical Erlenmeyer flask scale. This proof of concept was performed using the wild type strain and 23 randomly selected mutant strains. One specific mutant strain with an enhanced production behavior could be obtained.

Production of scopularide A in submerged culture with Scopulariopsis brevicaulis.

BACKGROUND: Marine organisms produce many novel compounds with useful biological activity, but are currently underexploited. Considerable research has been invested in the study of compounds from marine bacteria, and several groups have now recognised that marine fungi also produce an interesting range of compounds. During product discovery, these compounds are often produced only in non-agitated culture conditions, which are unfortunately not well suited for scaling up. A marine isolate of Scopulariopsis brevicaulis, strain LF580, produces the cyclodepsipeptide scopularide A, which has previously only been produced in non-agitated cultivation. RESULTS: Scopulariopsis brevicaulis LF580 produced scopularide A when grown in batch and fed-batch submerged cultures. Scopularide A was extracted primarily from the biomass, with approximately 7% being extractable from the culture supernatant. By increasing the biomass density of the cultivations, we were able to increase the volumetric production of the cultures, but it was important to avoid nitrogen limitation. Specific production also increased with increasing biomass density, leading to improvements in volumetric production up to 29-fold, compared with previous, non-agitated cultivations. Cell densities up to 36 g L-1 were achieved in 1 to 10 L bioreactors. Production of scopularide A was optimised in complex medium, but was also possible in a completely defined medium. CONCLUSIONS: Scopularide A production has been transferred from a non-agitated to a stirred tank bioreactor environment with an approximately 6-fold increase in specific and 29-fold increase in volumetric production. Production of scopularide A in stirred tank bioreactors demonstrates that marine fungal compounds can be suitable for scalable production, even with the native production organism.

Bioaccumulation and biovolatilization of various elements using filamentous fungus Scopulariopsis brevicaulis.

UNLABELLED: Biovolatilization and bioaccumulation capabilities of different elements by microscopic filamentous fungus Scopulariopsis brevicaulis were observed. Accumulation of As(III), As(V), Se(IV), Se(VI), Sb(III), Sb(V), Te(IV), Te(VI), Hg(II), Tl(I) and Bi(III) by S. brevicaulis was quantified by analysing the amount of elements in biomass of the fungus using ICP AAS. The highest amounts of bioaccumulated metal(loid)s were obtained as follows: Bi(III) > Te(IV) > Hg(II) > Se(IV) > Te(VI) > Sb(III) at different initial contents, with Bi(III) accumulation approximately 87%. The highest percentages of volatilization were found using Hg(II) (50%) and Se(IV) (46·5%); it was also demonstrated with all studied elements. This proved the biovolatilization ability of microscopic fungi under aerobic conditions. The highest removed amount was observed using Hg(II) (95·30%), and more than 80% of Se(IV), Te(IV), Bi(III) and Hg(II) was removed by bioaccumulation and biovolatilization, which implies the possibilities of use of these processes for bioremediations. There were reported significant differences between bioaccumulation and biovolatilization of almost all applied metal(loid)s if valence is mentioned. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial accumulation and volatilization are natural processes involved in biogeochemical cycles of elements. Despite their impact on mobility, bioavailability and toxicity of various metal(loid)s, only few papers deal with these processes under aerobic conditions with microscopic fungi. Thus, the proving of ability of microscopic fungus Scopulariopsis brevicaulis to accumulate and transform metals and metalloids by methylation or alkylation and quantification of these processes were demonstrated. The results can provide basic information on natural elements cycling and background for more specific studies focusing, for example, on application of these processes in mitigation of metal(loid) contamination.

Fungal contamination of poultry litter: a public health problem.

Although numerous studies have been conducted on microbial contaminants associated with various stages related to poultry and meat products processing, only a few reported on fungal contamination of poultry litter. The goals of this study were to (1) characterize litter fungal contamination and (2) report the incidence of keratinophilic and toxigenic fungi presence. Seven fresh and 14 aged litter samples were collected from 7 poultry farms. In addition, 27 air samples of 25 litters were also collected through impaction method, and after laboratory processing and incubation of collected samples, quantitative colony-forming units (CFU/m³) and qualitative results were obtained. Twelve different fungal species were detected in fresh litter and Penicillium was the most frequent genus found (59.9%), followed by Alternaria (17.8%), Cladosporium (7.1%), and Aspergillus (5.7%). With respect to aged litter, 19 different fungal species were detected, with Penicillium sp. the most frequently isolated (42.3%), followed by Scopulariopsis sp. (38.3%), Trichosporon sp. (8.8%), and Aspergillus sp. (5.5%). A significant positive correlation was found between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m³). Litter fungal quantification and species identification have important implications in the evaluation of potential adverse health risks to exposed workers and animals. Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genus) as well as toxigenic fungi (Aspergillus, Fusarium, and Penicillium genus) were isolated.