Degradation of Bunker C Fuel Oil by White-Rot Fungi in Sawdust Cultures Suggests Potential Applications in Bioremediation

<div><p>Fungal lignocellulolytic enzymes are promising agents for oxidizing pollutants. This study investigated degradation of Number 6 “Bunker C” fuel oil compounds by the white-rot fungi <i>Irpex lacteus</i>, <i>Trichaptum biforme</i>, <i>Phlebia radiata</i>, <i>Trametes versicolor</i>, and <i>Pleurotus ostreatus</i> (Basidiomycota, Agaricomycetes). Averaging across all studied species, 98.1%, 48.6%, and 76.4% of the initial Bunker C C10 alkane, C14 alkane, and phenanthrene, respectively were degraded after 180 days of fungal growth on pine media. This study also investigated whether Bunker C oil induces changes in gene expression in the white-rot fungus <i>Punctularia strigosozonata</i>, for which a complete reference genome is available. After 20 days of growth, a monokaryon <i>P</i>. <i>strigosozonata</i> strain degraded 99% of the initial C10 alkane in both pine and aspen media but did not affect the amounts of the C14 alkane or phenanthrene. Differential gene expression analysis identified 119 genes with ≥ log₂(2-fold) greater expression in one or more treatment comparisons. Six genes were significantly upregulated in media containing oil; these genes included three enzymes with potential roles in xenobiotic biotransformation. Carbohydrate metabolism genes showing differential expression significantly accumulated transcripts on aspen vs. pine substrates, perhaps reflecting white-rot adaptations to growth on hardwood substrates. The mechanisms by which <i>P</i>. <i>strigosozonata</i> may degrade complex oil compounds remain obscure, but degradation results of the 180-day cultures suggest that diverse white-rot fungi have promise for bioremediation of petroleum fuels.</p></div

Illumina RNA-Seq read counts and percentages of reads mapped to the <i>Punctularia strigosozonata</i> genome and transcriptome.

<p>Illumina RNA-Seq read counts and percentages of reads mapped to the <i>Punctularia strigosozonata</i> genome and transcriptome.</p

Biplot of principal components (PC) axes PC1 and PC2 derived from a Principal Components Analysis (PCA) of mapped paired-end Illumina RNA-Seq reads from 20 day-old <i>Punctularia strigosozonata</i> cultures in media treatments (aspen, aspen with Bunker C oil, pine, and pine with Bunker C oil).

<p>Ordination was completed in the R package DESeq.</p

<i>Punctularia strigosozonata</i> transcripts with predicted protein functions expressed ≥ log₂(2-fold) (adjusted p < 0.01) in comparisons of 20-day growth on aspen and pine media with and without Bunker C oil.

<p>Positive log₂ fold changes indicate transcript accumulation in the first treatment while negative log₂ fold changes indicate transcript accumulation in the second treatment.</p><p><i>Punctularia strigosozonata</i> transcripts with predicted protein functions expressed ≥ log₂(2-fold) (adjusted p < 0.01) in comparisons of 20-day growth on aspen and pine media with and without Bunker C oil.</p

Degradation (%) of phenanthrene, a C14 alkane, and a C10 alkane in Bunker C oil by white-rot fungi.

<p>Degradation percentages were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130381#pone.0130381.e001" target="_blank">Eq 1</a>.</p><p>Degradation (%) of phenanthrene, a C14 alkane, and a C10 alkane in Bunker C oil by white-rot fungi.</p

Hydrocarbon degradation by <i>Trichaptum biforme</i>.

<p>GC-MS chromatograms of (a) alkane and (b) phenanthrene degradation by <i>T</i>. <i>biforme</i> measured after 180 days of growth in pine media with Bunker C oil. Black lines = <i>T</i>. <i>biforme</i> profiles; blue lines = Bunker C oil profiles.</p

MOESM1 of Genome-wide analysis of cytochrome P450s of Trichoderma spp.: annotation and evolutionary relationships

Additional file 1. Table S1: List of Cyp protein entries with incomplete cytochrome P450 domain. Table S2: Cytochrome P450s associated with predicted secondary metabolism-related gene clusters

MOESM1 of The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa

Additional file 1: Figure S1. Schematic depiction of phylogeny and conserved domains/signals in the pdr-1 gene and its orthologs. The amino acid sequence of N. crassa PDR-1 (NCU09033), A. niger RhaR (An13g00910), A. nidulans RhaR (AN5673) and P. stipitis TRC1 (ABN68604) was used in a conserved domain search, as well as NLS and NES prediction. The phylogeny of these proteins was determined. B. cinerea GaaR (Bcin09g00170) constitutes the outgroup in the phylogenetic tree. GAL4 = GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA-binding domain, fungal TF MHR = fungal transcription factor regulatory middle homology region, green triangle = nuclear localization signal, red triangle = nuclear export signal. Figure S2. Growth phenotypes and protein secretion of N. crassa WT, Δpdr-1 and pdr-1-comp strains. (A) Observed growth phenotypes. Strains were grown on either 2 mM l-Rha, 2 mM d-Xyl, 1% pectin or 1% xylan. The cultures were incubated for 3 days. (B) Sucrose pregrown cultures were switched to pectin medium and the concentration of secreted protein was determined. Error bars represent standard deviation (n = 3). Significance was determined by an independent two-sample t-test of WT against Δpdr-1 or pdr-1-comp with *p < 0.05. Figure S3. Venn diagrams of DEseq results and correlation studies of RNA-seq to RT-qPCR data. Strains were pregrown for 16 h on 2% sucrose and then switched to an induction medium of either 1% pectin (pec) or 2 mM l-Rha for an additional 4 h. (A) Differential expression analysis (DEseq) was performed on the RNA-seq data. Genes of the WT and the Δpdr-1 strains that were threefold upregulated (left diagram; +) or downregulated (right diagram; −) were compared. Venn diagrams were created with: http://bioinformatics.psb.ugent.be/webtools/Venn/ . WT on 1% pectin was used in biological duplicates; all other conditions were used in biological triplicates for the RNA-seq analysis. (B) Correlation analysis of RT-qPCR data to RNA-seq data. Axes are log10-scaled. The fold change expression of several key pectinase genes was determined by RT-qPCR in the WT and Δpdr-1 strain grown on 1% pectin and plotted against their respective RNA-seq data. The Pearson’s correlation coefficient (ρ) was determined. Two biological replicates were used for RT-qPCR except for ply-2, where only one was used. All biological replicates were analyzed as three technical replicates. Figure S4. Expression levels of pdr-1, gh28-1 and NCU09034 determined by RT-qPCR. (A) The strains were grown for 2 days on 1% pectin and the expression level of pdr-1 in the pdr-1-oex strain or gh28-1 in the gh28-1-oex and gh28-1-comp strain was determined. The WT strain was used as reference. (B) The WT and pdr-1-oex strain were incubated for 48 h on 1% xylan. 2 mM l-Rha was added and the strains were incubated for an additional 30 min before harvesting the RNA for RT-qPCR. Strains incubated for the same time but without l-Rha were used as controls. The expression of pdr-1 and NCU09034 was determined for both strains. Three biological replicates were used, except for pdr-1 in WT on pectin and NCU09034 in WT on xylan plus l-Rha, where only two were used. All biological replicates were analyzed as three technical replicates. Error bars represent standard deviation. Significance was determined by an independent two-sample t-test with **p < 0.01, ***p < 0.001. Figure S5. Expression profile of pdr-1 and biomass accumulation of the pdr-1-oex strain. (A) Expression profile of pdr-1. After a 16 h pre-incubation on 2% sucrose, biomass of the WT strain was washed three times with 1× Vogel’s solution and transferred to a medium of either no carbon source (NoC), 2% sucrose, 2 mM l-Rha or 1% pectin. The strain was incubated for an additional 4 h. Error bars represent standard deviation (n = 2 for 1% pectin, n = 3 for NoC, 2% sucrose and 2 mM l-Rha). (B) Determination of accumulated biomass. Strains were grown on 1% xylan with 0.5 mM l-Rha or d-GalA. A medium containing 1% xylan was used as control. Biomass was determined by dry weight. Error bars represent standard deviation (n = 3)

Additional file 3: Figure S1. of Comparative genomics of Coniophora olivacea reveals different patterns of genome expansion in Boletales

Snapshot of synteny dot plot between C. olivacea and C. puteana. (TIFF 582 kb

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

27 juin 19371937/06/27 (A38,N13339)