Analysis of the microbiome of the Bolivian high-altitude Lake Pastos Grandes.

Lake Pastos Grandes in Bolivia is mainly composed of salt flats, which are sporadically and only partially submerged during the wet season. In the present study, the chemical composition of water samples of the lake and some influent rivers was determined. We found that it is likely that the lake was influenced by the dilution of metals from ancient evaporites. We performed the first metagenomic studies of this lake. Analyses of shotgun metagenomics revealed that the relative abundances of Burkholderiales and Pseudomonadales were noteworthy in the water samples, whereas the archaea belonging to the Halobacteriales and Cyanobacteria from subsection III had high abundances in the salt flat. The eukaryotes Crustacea and Diatomea exhibited the highest abundances in the water samples. We investigated further the potential effect of human activities on the nitrogen cycle mobilization in the lake and the propagation of antimicrobial resistance genes. This is the first report about the cycle in the lake. Additionally, rifamycin resistance genes and genes related to efflux pumps, which are not considered a hazard when identified in metagenomes, had the uppermost relative abundances in all sampling points. We found that Lake Pastos Grandes hitherto does not show an appreciable influence by anthropogenic actions.

Removal of total phosphorus, ammonia nitrogen and organic carbon from non-sterile municipal wastewater with Trametes versicolor and Aspergillus luchuensis.

Discharge of organic load from treated wastewater may cause environmental eutrophication. Recently, fungi have gained much attention due to their removal of pharmaceutical substances by enzymatic degradation and adsorption. However, the fungal effect in removing nutrients is less investigated. Therefore, two fungal species, the white-rot fungus T. versicolor as a laboratory strain and the mold A. luchuensis as an environmental isolate from the municipal wastewater treatment plant, were studied to determine the fungal potential for phosphorus, nitrogen, and the total organic carbon removal from municipal wastewater, carrying out a batch scale experiment to a fluidized bed pelleted bioreactor. During the batch scale experiment, the total removal (99.9 %) of phosphorus by T. versicolor was attained after a 6 h-long incubation period while the maximal removal efficiency (99.9 %) for phosphorus from A. luchuensis was gained after an incubation period of 24 h. Furthermore, both fungi showed that the pH adjustment to 5.5 kept the concentration of nitrogen constant and stabilized the total organic carbon reduction process for the entire incubation period. The results from the fluidized bed bioreactor demonstrated opposite tendencies on a nutrient removal comparing to a batch experiment where no significant effect on phosphorus, nitrogen, and total organics carbon reduction was observed. The obtained results from this study of batch and fluidized bed bioreactor experiments are a promising starting point for a successful fungal treatment optimization and application to wastewater treatment.

Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides.

Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.

Antifeedants Produced by Bacteria Associated with the Gut of the Pine Weevil Hylobius abietis.

The pine weevil, Hylobius abietis, is a severe forest pest insect as it feeds on newly planted conifer seedlings. To identify and develop an antifeedant could be one step towards the protection of seedlings from feeding damage by the pine weevil. With the aim to trace the origin of the antifeedants previously found in feces of the pine weevil, we investigated the culturable bacteria associated with the gut and identified the volatiles they produced. Bacterial isolates were identified by 16S ribosomal RNA gene analysis. The volatile emissions of selected bacteria, cultivated on NB media or on the grated phloem of Scots pine twigs dispersed in water, were collected and analyzed by solid-phase microextraction gas chromatography-mass spectrometry. The bacterial isolates released a variety of compounds, among others 2-methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol, 1-octen-3-ol, 3-octanone, dimethyl disulfide, and dimethyl trisulfide. A strong antifeedant effect was observed by 2-phenylethanol, which could thus be a good candidate for use to protect planted conifer seedlings against feeding damage caused by H. abietis.

Cedrol, a malaria mosquito oviposition attractant is produced by fungi isolated from rhizomes of the grass Cyperus rotundus.

BACKGROUND: Cedrol, a sesquiterpene alcohol, is the first identified oviposition attractant for African malaria vectors. Finding the natural source of this compound might help to elucidate why Anopheles gambiae and Anopheles arabiensis prefer to lay eggs in habitats containing it. Previous studies suggest that cedrol may be a fungal metabolite and the essential oil of grass rhizomes have been described to contain a high amount of different sesquiterpenes. RESULTS: Rhizomes of the grass Cyperus rotundus were collected in a natural malaria mosquito breeding site. Two fungi were isolated from an aqueous infusion with these rhizomes. They were identified as Fusarium falciforme and a species in the Fusarium fujikuroi species complex. Volatile compounds were collected from the headspace above fungal cultures on Tenax traps which were analysed by gas chromatography-mass spectrometry (GCMS). Cedrol and a cedrol isomer were detected in the headspace above the F. fujikuroi culture, while only cedrol was detected above the F. falciforme culture. CONCLUSION: Cedrol an oviposition attractant for African malaria vectors is produced by two fungi species isolated from grass rhizomes collected from a natural mosquito breeding site.

Chemodiversity and biodiversity of fungi associated with the pine weevil Hylobius abietis.

The pine weevil Hylobius abietis is a severe pest of conifer seedlings in reforestation areas. Weevils lay eggs in the root bark or in the soil near roots of recently dead trees and cover the eggs with frass (feces combined with chewed bark), possibly to avoid conspecific egg predation. The aim of the present investigation focused on isolation, identification, and volatile production of fungi from pine-weevil feces and frass. Fungi were isolated from weevil frass and feces separately, followed by identification based on ITS sequencing. Fifty-nine isolates belonging to the genera Penicillium, Ophiostoma, Mucor, Leptographium, Eucasphaeria, Rhizosphaera, Debaryomyces, and Candida were identified. Volatile organic compounds (VOCs) produced by the fungal community and fungal isolates cultured on weevil-frass broth were identified by SPME-GCMS. Major VOCs emitted from the fungal community and pure isolates were species- and strain specific and included isopentylalcohol, styrene, 3-octanone, 6-protoilludene, methyl salicylate, 3-methylanisole, 2-methoxyphenol, and phenol. Some of these are known to influence the orientation of pine weevils when tested among highly attractive newly planted conifer seedlings.

Sustainable bio-production of styrene from forest waste.

A strain of Penicillium expansum was studied for the production of styrene using forest waste biomass as a feeding substrate. The fungal strain was cultivated on bark of various trees supplemented with yeast extract and the volatiles produced were collected on Tenax TA and analyzed by gas chromatography-mass spectrometry. Fungus cultured on grated soft bark of pine (Pinus sylvestris) stems (GPB) and mature bark of oak (Quercus robur) supplemented with yeast extract produced relatively the highest amounts of styrene. The maximum styrene production rate was 52.5 µg/h, 41 µg/h and 27 µg/h from fungus cultivated on 50 mL liquid media with 10 g GPB or mature bark of oak and potato dextrose broth respectively. These promising results suggest that the fungal strain could be used to produce "green" styrene plastics using renewable forest waste biomass.

Efficacy of reactive mineral-based sorbents for phosphate, bacteria, nitrogen and TOC removal--column experiment in recirculation batch mode.

Two mineral-based materials (Polonite and Sorbulite) intended for filter wells in on-site wastewater treatment were compared in terms of removal of phosphate (PO4-P), total inorganic nitrogen (TIN), total organic carbon (TOC) and faecal indicator bacteria (Escherichia coli and Enterococci). Using an innovative, recirculating system, septic tank effluent was pumped at a hydraulic loading rate of 3000 L m(2) d(-1) into triplicate bench-scale columns of each material over a 90-day period. The results showed that Polonite performed better with respect to removal of PO4-P, retaining on average 80% compared with 75% in Sorbulite. This difference was attributed to higher CaO content in Polonite and its faster dissolution. Polonite also performed better in terms of removal of bacteria because of its higher pH value. The total average reduction in E. coli was 60% in Polonite and 45% in Sorbulite, while for Enterococci the corresponding value was 56% in Polonite and 34% in Sorbulite. Sorbulite removed TIN more effectively, with a removal rate of 23%, while Polonite removed 11% of TIN, as well as TOC. Organic matter (measured as TOC) was accumulated in the filter materials but was also released periodically. The results showed that Sorbulite could meet the demand in removing phosphate and nitrogen with reduced microbial release from the wastewater treatment process.

Penicillium expansum volatiles reduce pine weevil attraction to host plants.

The pine weevil Hylobius abietis (L.) is a severe pest of conifer seedlings in reforested areas of Europe and Asia. To identify minimally toxic and ecologically sustainable compounds for protecting newly planted seedlings, we evaluated the volatile metabolites produced by microbes isolated from H. abietis feces and frass. Female weevils deposit feces and chew bark at oviposition sites, presumably thus protecting eggs from feeding conspecifics. We hypothesize that microbes present in feces/frass are responsible for producing compounds that deter weevils. Here, we describe the isolation of a fungus from feces and frass of H. abietis and the biological activity of its volatile metabolites. The fungus was identified by morphological and molecular methods as Penicillium expansum Link ex. Thom. It was cultured on sterilized H. abietis frass medium in glass flasks, and volatiles were collected by SPME and analyzed by GC-MS. The major volatiles of the fungus were styrene and 3-methylanisole. The nutrient conditions for maximum production of styrene and 3-methylanisole were examined. Large quantities of styrene were produced when the fungus was cultured on grated pine bark with yeast extract. In a multi-choice arena test, styrene significantly reduced male and female pine weevils' attraction to cut pieces of Scots pine twigs, whereas 3-methylanisole only reduced male weevil attraction to pine twigs. These studies suggest that metabolites produced by microbes may be useful as compounds for controlling insects, and could serve as sustainable alternatives to synthetic insecticides.

Cell entry and antimicrobial properties of eukaryotic cell-penetrating peptides.

Antimicrobial drug action is limited by both microbial and host cell membranes. Microbes stringently exclude the entry of most drugs, and mammalian membranes limit drug distribution and access to intracellular pathogens. Recently, cell-penetrating peptides (CPPs) have been developed as carriers to improve mammalian cell uptake. Given that CPPs are cationic and often amphipathic, similar to membrane active antimicrobial peptides, it may be possible to use CPP activity to improve drug delivery to microbes. Here, two CPPs, TP10 and pVEC, were found to enter a range of bacteria and fungi. The uptake route involves rapid surface accumulation within minutes followed by cell entry. TP10 inhibited Candida albicans and Staphylococcus aureus growth, and pVEC inhibited Mycobacterium smegmatis growth at low micromolar doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damage microbes, and this effect was sufficient to clear HeLa cell cultures from noninvasive S. aureus infection. Also, conversion of the cytotoxicity indicator dye SYTOX Green showed that TP10 causes rapid and lethal permeabilization of S. aureus and pVEC permeabilizes M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC can enter both mammalian and microbial cells and preferentially permeabilize and kill microbes.

The signal peptide NPFSD fused to ricin A chain enhances cell uptake and cytotoxicity in Candida albicans.

Microorganisms possess stringent cell membranes which limit the cellular uptake of antimicrobials. One strategy to overcome these barriers is to attach drugs or research reagents to carrier peptides that enter cells by passive permeation or active uptake. Here the short endocytosis signal peptide NPFSD was found to efficiently deliver both FITC and GFP into Saccharomyces cerevisiae and Candida albicans with uptake into the majority of cells in a population. The NPFSD signal is itself non-toxic, but when fused to the ricin A chain toxin (RTA) the peptide enhanced both cell uptake and toxicity against C. albicans, which like other yeasts is resistant to naked RTA. Cell entry required at least 1 h incubation, temperatures above 4 degrees C, and an energy source, and uptake was out-competed with free peptide. Therefore, the NPFSD peptide can carry a range of compounds into yeasts and this delivery route holds promise to enhance the activity of antifungals.

Peptide-mediated delivery of green fluorescent protein into yeasts and bacteria.

Stringent microbial cell barriers limit the application of many substances in research and therapeutics. Carrier peptides that penetrate or translocate across cell membranes may help overcome this problem. To assess peptide-mediated delivery into two yeast and three bacterial species, a range of cell penetrating and signal peptide sequences were fused to green fluorescent protein (GFP), expressed in Escherichia coli, partially purified and incubated with growing cells. Fluorescence microscopy indicated several peptides that mediated delivery. In particular, VLTNENPFSDP efficiently delivered GFP into Candida albicans and Staphylococcus aureus, while YKKSNNPFSD was most efficient for Bacillus subtilis and CFFKDEL for Escherichia coli. Carrier peptides may improve delivery of certain large molecular mass molecules into microorganisms for research and therapeutic applications.