Isolation, identification, and biochemical characterization of a novel bifunctional phosphomannomutase/phosphoglucomutase from the metagenome of the brown alga Laminaria digitata.

Macroalgae host diverse epiphytic bacterial communities with potential symbiotic roles including important roles influencing morphogenesis and growth of the host, nutrient exchange, and protection of the host from pathogens. Macroalgal cell wall structures, exudates, and intra-cellular environments possess numerous complex and valuable carbohydrates such as cellulose, hemi-cellulose, mannans, alginates, fucoidans, and laminarin. Bacterial colonizers of macroalgae are important carbon cyclers, acquiring nutrition from living macroalgae and also from decaying macroalgae. Seaweed epiphytic communities are a rich source of diverse carbohydrate-active enzymes which may have useful applications in industrial bioprocessing. With this in mind, we constructed a large insert fosmid clone library from the metagenome of Laminaria digitata (Ochrophyta) in which decay was induced. Subsequent sequencing of a fosmid clone insert revealed the presence of a gene encoding a bifunctional phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme 10L6AlgC, closely related to a protein from the halophilic marine bacterium, Cobetia sp. 10L6AlgC was subsequently heterologously expressed in Escherichia coli and biochemically characterized. The enzyme was found to possess both PMM and PGM activity, which had temperature and pH optima of 45°C and 8.0, respectively; for both activities. The PMM activity had a K m of 2.229 mM and V max of 29.35 mM min-1 mg-1, while the PGM activity had a K m of 0.5314 mM and a V max of 644.7 mM min-1 mg-1. Overall characterization of the enzyme including the above parameters as well as the influence of various divalent cations on these activities revealed that 10L6AlgC has a unique biochemical profile when compared to previously characterized PMM/PGM bifunctional enzymes. Thus 10L6AlgC may find utility in enzyme-based production of biochemicals with different potential industrial applications, in which other bacterial PMM/PGMs have previously been used such as in the production of low-calorie sweeteners in the food industry.

Identification of BgP, a Cutinase-Like Polyesterase From a Deep-Sea Sponge-Derived Actinobacterium.

Many marine bacteria produce extracellular enzymes that degrade complex molecules to facilitate their growth in environmental conditions that are often harsh and low in nutrients. Marine bacteria, including those inhabiting sea sponges, have previously been reported to be a promising source of polyesterase enzymes, which have received recent attention due to their potential ability to degrade polyethylene terephthalate (PET) plastic. During the screening of 51 marine bacterial isolates for hydrolytic activities targeting ester and polyester substrates, a Brachybacterium ginsengisoli B129SM11 isolate from the deep-sea sponge Pheronema sp. was identified as a polyesterase producer. Sequence analysis of genomic DNA from strain B129SM11, coupled with a genome "mining" strategy, allowed the identification of potential polyesterases, using a custom database of enzymes that had previously been reported to hydrolyze PET or other synthetic polyesters. This resulted in the identification of a putative PET hydrolase gene, encoding a polyesterase-type enzyme which we named BgP that shared high overall similarity with three well-characterized PET hydrolases-LCC, TfCut2, and Cut190, all of which are key enzymes currently under investigation for the biological recycling of PET. In silico protein analyses and homology protein modeling offered structural and functional insights into BgP, and a detailed comparison with Cut190 revealed highly conserved features with implications for both catalysis and substrate binding. Polyesterase activity was confirmed using an agar-based polycaprolactone (PCL) clearing assay, following heterologous expression of BgP in Escherichia coli. This is the first report of a polyesterase being identified from a deep-sea sponge bacterium such as Brachybacterium ginsengisoli and provides further insights into marine-derived polyesterases, an important family of enzymes for PET plastic hydrolysis. Microorganisms living in association with sponges are likely to have increased exposure to plastics and microplastics given the wide-scale contamination of marine ecosystems with these plastics, and thus they may represent a worthwhile source of enzymes for use in new plastic waste management systems. This study adds to the growing knowledge of microbial polyesterases and endorses further exploration of marine host-associated microorganisms as a potentially valuable source of this family of enzymes for PET plastic hydrolysis.

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation.

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics.

Diversity of bacteria populations associated with different thallus regions of the brown alga Laminaria digitata.

Stipitate kelp species such as Laminaria digitata dominate most cold-water subtidal rocky shores and form underwater forests which are among the most productive coastal systems worldwide. Laminaria also sustains rich bacterial communities which offer a variety of biotechnological applications. However, to date, in-depth studies on the diversity and uniqueness of bacterial communities associated with this macroalgal species, their ecological role and their interactions with the alga are under-represented. To address this, the epibacterial populations associated with different thallus regions (holdfast, stipe, meristem, blade) of this brown seaweed were investigated using high-throughput Illumina sequencing of the 16S rRNA genes. The results show that epibacterial communities of the brown seaweed are significantly different and specific to the thallus region, with the shared bacterial population comprising of only 1.1% of the total amplicon sequence variants. The diverse holdfast and blade tissues formed distinct clusters while the meristem and stipe regions are more closely related. The data obtained further supports the hypothesis that macroalgal bacterial communities are shaped by morphological niches and display specificity.

Corrigendum: Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

[This corrects the article DOI: 10.3389/fmicb.2019.01713.].

In silico Screening and Heterologous Expression of a Polyethylene Terephthalate Hydrolase (PETase)-Like Enzyme (SM14est) With Polycaprolactone (PCL)-Degrading Activity, From the Marine Sponge-Derived Strain Streptomyces sp. SM14.

Plastics, such as the polyethylene terephthalate (PET), are widely used for various industrial applications, due to their physicochemical properties which are particularly useful in the packaging industry. However, due to improper plastic waste management and difficulties in recycling, post-consumer plastic waste has become a pressing issue for both the environment and for human health. Hence, novel technologies and methods of processing plastic waste are required to address these issues. Enzymatic-assisted hydrolysis of synthetic polymers has been proposed as a potentially more efficient and environment-friendly alternative to the currently employed methods. Recently, a number of PET hydrolases have been described, and in particular a PETase derived from Ideonella sakaiensis 201-F6 (IsPETase), which appears to be the most efficient and substrate-specific bacterial PET hydrolase enzyme discovered to date. In order to further investigate this class of PETase-like enzymes, we employed an in silico-based screening approach on the biotechnologically relevant genus Streptomyces, including terrestrial and marine isolates; in a search for potential PETase homologs. From a total of 52 genomes analyzed, we were able to identify three potential PETase-like enzymes, all of which were derived from marine-sponge associated Streptomyces isolates. A candidate PETase-like gene (SM14est) was identified in Streptomyces sp. SM14. Further in silico characterization of the SM14est protein sequence and its predicted three-dimensional structure were performed and compared to the well-characterized IsPETase. Both the serine hydrolase motif Gly-x1-Ser-x2-Gly and the catalytic triad Ser, Asp, His are conserved in both sequences. Molecular docking experiments indicated that the SM14est enzyme possessed the capacity to bind plastics as substrates. Finally, polyesterase activity was confirmed using a polycaprolactone (PCL) plate clearing assay which is a model substrate for the degradation of plastics; following heterologous expression of SM14est in Escherichia coli, with secretion being facilitated by the native Streptomyces signal peptide. These findings provide further insights into this important class of PETase-like enzymes.

Genome Mining Coupled with OSMAC-Based Cultivation Reveal Differential Production of Surugamide A by the Marine Sponge Isolate Streptomyces sp. SM17 When Compared to Its Terrestrial Relative S. albidoflavus J1074.

Much recent interest has arisen in investigating Streptomyces isolates derived from the marine environment in the search for new bioactive compounds, particularly those found in association with marine invertebrates, such as sponges. Among these new compounds recently identified from marine Streptomyces isolates are the octapeptidic surugamides, which have been shown to possess anticancer and antifungal activities. By employing genome mining followed by an one strain many compounds (OSMAC)-based approach, we have identified the previously unreported capability of a marine sponge-derived isolate, namely Streptomyces sp. SM17, to produce surugamide A. Phylogenomics analyses provided novel insights on the distribution and conservation of the surugamides biosynthetic gene cluster (sur BGC) and suggested a closer relatedness between marine-derived sur BGCs than their terrestrially derived counterparts. Subsequent analysis showed differential production of surugamide A when comparing the closely related marine and terrestrial isolates, namely Streptomyces sp. SM17 and Streptomyces albidoflavus J1074. SM17 produced higher levels of surugamide A than S. albidoflavus J1074 under all conditions tested, and in particular producing >13-fold higher levels when grown in YD and 3-fold higher levels in SYP-NaCl medium. In addition, surugamide A production was repressed in TSB and YD medium, suggesting that carbon catabolite repression (CCR) may influence the production of surugamides in these strains.

Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

The emergence of antibiotic resistant microorganisms has led to an increased need for the discovery and development of novel antimicrobial compounds. Frequent rediscovery of the same natural products (NPs) continues to decrease the likelihood of the discovery of new compounds from soil bacteria. Thus, efforts have shifted toward investigating microorganisms and their secondary metabolite biosynthesis potential, from diverse niche environments, such as those isolated from marine sponges. Here we investigated at the genomic level two Streptomyces spp. strains, namely SM17 and SM18, isolated from the marine sponge Haliclona simulans, with previously reported antimicrobial activity against clinically relevant pathogens; using single molecule real-time (SMRT) sequencing. We performed a series of comparative genomic analyses on SM17 and SM18 with their closest terrestrial relatives, namely S. albus J1074 and S. pratensis ATCC 33331 respectively; in an effort to provide further insights into potential environmental niche adaptations (ENAs) of marine sponge-associated Streptomyces, and on how these adaptations might be linked to their secondary metabolite biosynthesis potential. Prediction of secondary metabolite biosynthetic gene clusters (smBGCs) indicated that, even though the marine isolates are closely related to their terrestrial counterparts at a genomic level; they potentially produce different compounds. SM17 and SM18 displayed a better ability to grow in high salinity medium when compared to their terrestrial counterparts, and further analysis of their genomes indicated that they possess a pool of 29 potential ENA genes that are absent in S. albus J1074 and S. pratensis ATCC 33331. This ENA gene pool included functional categories of genes that are likely to be related to niche adaptations and which could be grouped based on potential biological functions such as osmotic stress, defense; transcriptional regulation; symbiotic interactions; antimicrobial compound production and resistance; ABC transporters; together with horizontal gene transfer and defense-related features.

Biochemical Characterization of a Novel Monospecific Endo-ß-1,4-Glucanase Belonging to GH Family 5 From a Rhizosphere Metagenomic Library.

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-ß-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modeling analysis indicated that CelRH5 is related to endo-ß-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum, and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC), and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5-8.5. Highest activity was observed at 40°C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30°C. The presence of the final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+, and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulfate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications.

Microbial Population Changes in Decaying Ascophyllum nodosum Result in Macroalgal-Polysaccharide-Degrading Bacteria with Potential Applicability in Enzyme-Assisted Extraction Technologies.

Seaweeds are of significant interest in the food, pharmaceutical, and agricultural industries as they contain several commercially relevant bioactive compounds. Current extraction methods for macroalgal-derived metabolites are, however, problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods, such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25 °C, and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina sequencing and culture-dependent approaches, including the novel ichip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculosus and were shown to be more efficient than commercial enzyme preparations in their extraction efficiencies.

Graphene Facilitates Biomethane Production from Protein-Derived Glycine in Anaerobic Digestion.

Interspecies electron transfer is a fundamental factor determining the efficiency of anaerobic digestion (AD), which involves syntrophy between fermentative bacteria and methanogens. Direct interspecies electron transfer (DIET) induced by conductive materials can optimize this process offering a significant improvement over indirect electron transfer. Herein, conductive graphene was applied in the AD of protein-derived glycine to establish DIET. The electron-producing reaction via DIET is thermodynamically more favorable and exhibits a more negative Gibbs free energy value (-60.0 kJ/mol) than indirect hydrogen transfer (-33.4 kJ/mol). The Gompertz model indicated that the kinetic parameters exhibited linear correlations with graphene addition from 0.25 to 1.0 g/L, leading to the highest increase in peak biomethane production rate of 28%. Sedimentibacter (7.8% in abundance) and archaea Methanobacterium (71.1%) and Methanosarcina (11.3%) might be responsible for DIET. This research can open up DIET to a range of protein-rich substrates, such as algae.

Extending the "One Strain Many Compounds" (OSMAC) Principle to Marine Microorganisms.

Genomic data often highlights an inconsistency between the number of gene clusters identified using bioinformatic approaches as potentially producing secondary metabolites and the actual number of chemically characterized secondary metabolites produced by any given microorganism. Such gene clusters are generally considered as "silent", meaning that they are not expressed under laboratory conditions. Triggering expression of these "silent" clusters could result in unlocking the chemical diversity they control, allowing the discovery of novel molecules of both medical and biotechnological interest. Therefore, both genetic and cultivation-based techniques have been developed aimed at stimulating expression of these "silent" genes. The principles behind the cultivation based approaches have been conceptualized in the "one strain many compounds" (OSMAC) framework, which underlines how a single strain can produce different molecules when grown under different environmental conditions. Parameters such as, nutrient content, temperature, and rate of aeration can be easily changed, altering the global physiology of a microbial strain and in turn significantly affecting its secondary metabolism. As a direct extension of such approaches, co-cultivation strategies and the addition of chemical elicitors have also been used as cues to activate "silent" clusters. In this review, we aim to provide a focused and comprehensive overview of these strategies as they pertain to marine microbes. Moreover, we underline how changes in some parameters which have provided important results in terrestrial microbes, but which have rarely been considered in marine microorganisms, may represent additional strategies to awaken "silent" gene clusters in marine microbes. Unfortunately, the empirical nature of the OSMAC approach forces scientists to perform extensive laboratory experiments. Nevertheless, we believe that some computation and experimental based techniques which are used in other disciplines, and which we discuss; could be effectively employed to help streamline the OSMAC based approaches. We believe that natural products discovery in marine microorganisms would be greatly aided through the integration of basic microbiological approaches, computational methods, and technological innovations, thereby helping unearth much of the as yet untapped potential of these microorganisms.

Diverse and Abundant Secondary Metabolism Biosynthetic Gene Clusters in the Genomes of Marine Sponge Derived Streptomyces spp. Isolates.

The genus Streptomyces produces secondary metabolic compounds that are rich in biological activity. Many of these compounds are genetically encoded by large secondary metabolism biosynthetic gene clusters (smBGCs) such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) which are modular and can be highly repetitive. Due to the repeats, these gene clusters can be difficult to resolve using short read next generation datasets and are often quite poorly predicted using standard approaches. We have sequenced the genomes of 13 Streptomyces spp. strains isolated from shallow water and deep-sea sponges that display antimicrobial activities against a number of clinically relevant bacterial and yeast species. Draft genomes have been assembled and smBGCs have been identified using the antiSMASH (antibiotics and Secondary Metabolite Analysis Shell) web platform. We have compared the smBGCs amongst strains in the search for novel sequences conferring the potential to produce novel bioactive secondary metabolites. The strains in this study recruit to four distinct clades within the genus Streptomyces. The marine strains host abundant smBGCs which encode polyketides, NRPS, siderophores, bacteriocins and lantipeptides. The deep-sea strains appear to be enriched with gene clusters encoding NRPS. Marine adaptations are evident in the sponge-derived strains which are enriched for genes involved in the biosynthesis and transport of compatible solutes and for heat-shock proteins. Streptomyces spp. from marine environments are a promising source of novel bioactive secondary metabolites as the abundance and diversity of smBGCs show high degrees of novelty. Sponge derived Streptomyces spp. isolates appear to display genomic adaptations to marine living when compared to terrestrial strains.

Synthesis of Nm-PHB (nanomelanin-polyhydroxy butyrate) nanocomposite film and its protective effect against biofilm-forming multi drug resistant Staphylococcus aureus.

Melanin is a dark brown ubiquitous photosynthetic pigment which have many varied and ever expanding applications in fabrication of radio-protective materials, food packaging, cosmetics and in medicine. In this study, melanin production in a Pseudomonas sp. which was isolated from the marine sponge Tetyrina citirna was optimized employing one-factor at a time experiments and characterized for chemical nature and stability. Following sonication nucleated nanomelanin (Nm) particles were formed and evaluated for antibacterial and antioxidant properties. Nanocomposite film was fabricated using combinations (% w/v) of polyhydroxy butyrate-nanomelanin (PHB:Nm) blended with 1% glycerol. The Nm was found to be spherical in shape with a diameter of 100-140 nm and showed strong antimicrobial activity against both Gram positive and Gram negative bacteria. The Nm-PHB nanocomposite film was homogeneous, smooth, without any cracks, and flexible. XRD and DSC data indicated that the film was crystalline in nature, and was thermostable up to 281.87 °C. This study represents the first report on the synthesis of Nm and fabrication of Nm-PHB nanocomposite film which show strong protective effect against multidrug resistant Staphyloccoccus aureus. Thus this Nm-PHB nanocomposite film may find utility as packaging material for food products by protecting the food products from oxidation and bacterial contamination.

Biotechnological Potential of Cold Adapted Pseudoalteromonas spp. Isolated from 'Deep Sea' Sponges.

The marine genus Pseudoalteromonas is known for its versatile biotechnological potential with respect to the production of antimicrobials and enzymes of industrial interest. We have sequenced the genomes of three Pseudoalteromonas sp. strains isolated from different deep sea sponges on the Illumina MiSeq platform. The isolates have been screened for various industrially important enzymes and comparative genomics has been applied to investigate potential relationships between the isolates and their host organisms, while comparing them to free-living Pseudoalteromonas spp. from shallow and deep sea environments. The genomes of the sponge associated Pseudoalteromonas strains contained much lower levels of potential eukaryotic-like proteins which are known to be enriched in symbiotic sponge associated microorganisms, than might be expected for true sponge symbionts. While all the Pseudoalteromonas shared a large distinct subset of genes, nonetheless the number of unique and accessory genes is quite large and defines the pan-genome as open. Enzymatic screens indicate that a vast array of enzyme activities is expressed by the isolates, including ß-galactosidase, ß-glucosidase, and protease activities. A ß-glucosidase gene from one of the Pseudoalteromonas isolates, strain EB27 was heterologously expressed in Escherichia coli and, following biochemical characterization, the recombinant enzyme was found to be cold-adapted, thermolabile, halotolerant, and alkaline active.

Simple screening protocol for identification of potential mycoremediation tools for the elimination of polycyclic aromatic hydrocarbons and phenols from hyperalkalophile industrial effluents.

A number of fungal strains belonging to the ascomycota, basidiomycota and zygomycota genera were subjected to an in vitro screening regime to assess their ligninolytic activity potential, with a view to their potential use in mycoremediation-based strategies to remove phenolic compounds and polycyclic aromatic hydrocarbons (PAHs) from industrial wastewaters. All six basidiomycetes completely decolorized remazol brilliant blue R (RBBR), while also testing positive in both the guaiacol and gallic acid tests indicating good levels of lignolytic activity. All the fungi were capable of tolerating phenanthrene, benzo-α- pyrene, phenol and p-chlorophenol in agar medium at levels of 10 ppm. Six of the fungal strains, Pseudogymnoascus sp., Aspergillus caesiellus, Trametes hirsuta IBB 450, Phanerochate chrysosporium ATCC 787, Pleurotus ostreatus MTCC 1804 and Cadophora sp. produced both laccase and Mn peroxidase activity in the ranges of 200-560 U/L and 6-152 U/L, respectively, in liquid media under nitrogen limiting conditions. The levels of adsorption of the phenolic and PAHs were negligible with 99% biodegradation being observed in the case of benzo-α-pyrene, phenol and p-chlorophenol. The aforementioned six fungal strains were also found to be able to effectively treat highly alkaline industrial wastewater (pH 12.4). When this wastewater was supplemented with 0.1 mM glucose, all of the tested fungi, apart from A. caesiellus, displayed the capacity to remove both the phenolic and PAH compounds. Based on their biodegradative capacity we found T. hirsuta IBB 450 and Pseudogymnoascus sp., to have the greatest potential for further use in mycoremediation based strategies to treat wastestreams containing phenolics and PAHs.

Characterization of lignocellulolytic activities from fungi isolated from the deep-sea sponge Stelletta normani.

Extreme habitats have usually been regarded as a source of microorganisms that possess robust proteins that help enable them to survive in such harsh conditions. The deep sea can be considered an extreme habitat due to low temperatures (<5°C) and high pressure, however marine sponges survive in these habitats. While bacteria derived from deep-sea marine sponges have been studied, much less information is available on fungal biodiversity associated with these sponges. Following screening of fourteen fungi isolated from the deep-sea sponge Stelletta normani sampled at a depth of 751 metres, three halotolerant strains (TS2, TS11 and TS12) were identified which displayed high CMCase and xylanase activities. Molecular based taxonomic approaches identified these strains as Cadophora sp. TS2, Emericellopsis sp. TS11 and Pseudogymnoascus sp. TS 12. These three fungi displayed psychrotolerance and halotolerant growth on CMC and xylan as sole carbon sources, with optimal growth rates at 20°C. They produced CMCase and xylanase activities, which displayed optimal temperature and pH values of between 50-70°C and pH 5-8 respectively, together with good thermostability and halotolerance. In solid-state fermentations TS2, TS11 and TS12 produced CMCases, xylanases and peroxidase/phenol oxidases when grown on corn stover and wheat straw. This is the first time that CMCase, xylanase and peroxidase/phenol oxidase activities have been reported in these three fungal genera isolated from a marine sponge. Given the biochemical characteristics of these ligninolytic enzymes it is likely that they may prove useful in future biomass conversion strategies involving lignocellulosic materials.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety.

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa.

Study of the performance of a thermophilic biological methanation system.

This study investigated the operation of ex-situ biological methanation at two thermophilic temperatures (55°C and 65°C). Methane composition of 85-88% was obtained and volumetric productivities of 0.45 and 0.4LCH4/Lreactor were observed at 55°C and 65°C after 24h respectively. It is postulated that at 55°C the process operated as a mixed culture as the residual organic substrates in the starting inoculum were still available. These were consumed prior to the assessment at 65°C; thus the methanogens were now dependent on gaseous substrates CO2 and H2. The experiment was repeated at 65°C with fresh inoculum (a mixed culture); methane composition and volumetric productivity of 92% and 0.46LCH4/Lreactor were achieved in 24h. Methanothermobacter species represent likely and resilient candidates for thermophilic biogas upgrading.

Diversity of Natural Product Biosynthetic Genes in the Microbiome of the Deep Sea Sponges Inflatella pellicula, Poecillastra compressa, and Stelletta normani.

Three different deep sea sponge species, Inflatella pellicula, Poecillastra compressa, and Stelletta normani comprising seven individual samples, retrieved from depths of 760-2900 m below sea level, were investigated using 454 pyrosequencing for their secondary metabolomic potential targeting adenylation domain and ketosynthase domain sequences. The data obtained suggest a diverse microbial origin of nonribosomal peptide synthetases and polyketide synthase fragments that in part correlates with their respective microbial community structures that were previously described and reveals an untapped source of potential novelty. The sequences, especially the ketosynthase fragments, display extensive clade formations which are clearly distinct from sequences hosted in public databases, therefore highlighting the potential of the microbiome of these deep sea sponges to produce potentially novel small-molecule chemistry. Furthermore, sequence similarities to gene clusters known to be involved in the production of many classes of antibiotics and toxins including lipopeptides, glycopeptides, macrolides, and hepatotoxins were also identified.