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

Population Level Analysis of Evolved Mutations Underlying Improvements in Plant Hemicellulose and Cellulose Fermentation by <i>Clostridium phytofermentans</i>

<div><p>Background</p><p>The complexity of plant cell walls creates many challenges for microbial decomposition. <i>Clostridium phytofermentans</i>, an anaerobic bacterium isolated from forest soil, directly breaks down and utilizes many plant cell wall carbohydrates. The objective of this research is to understand constraints on rates of plant decomposition by <i>Clostridium phytofermentans</i> and identify molecular mechanisms that may overcome these limitations.</p><p>Results</p><p>Experimental evolution via repeated serial transfers during exponential growth was used to select for <i>C. phytofermentans</i> genotypes that grow more rapidly on cellobiose, cellulose and xylan. To identify the underlying mutations an average of 13,600,000 paired-end reads were generated per population resulting in ∼300 fold coverage of each site in the genome. Mutations with allele frequencies of 5% or greater could be identified with statistical confidence. Many mutations are in carbohydrate-related genes including the promoter regions of glycoside hydrolases and amino acid substitutions in ABC transport proteins involved in carbohydrate uptake, signal transduction sensors that detect specific carbohydrates, proteins that affect the export of extracellular enzymes, and regulators of unknown specificity. Structural modeling of the ABC transporter complex proteins suggests that mutations in these genes may alter the recognition of carbohydrates by substrate-binding proteins and communication between the intercellular face of the transmembrane and the ATPase binding proteins.</p><p>Conclusions</p><p>Experimental evolution was effective in identifying molecular constraints on the rate of hemicellulose and cellulose fermentation and selected for putative gain of function mutations that do not typically appear in traditional molecular genetic screens. The results reveal new strategies for evolving and engineering microorganisms for faster growth on plant carbohydrates.</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

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

<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

Major fermentation product formation by cellulose adapted populations and founder after 10 days of growth.

<p>Major fermentation product formation by cellulose adapted populations and founder after 10 days of growth.</p

Growth, cellobiose utilization and ethanol production of cellobiose adapted populations and the founder.

<p>Growth (A) was measured every four hours as change in optical density in a spectrophotometer. Supernatant was collected every eight hours for measuring cellobiose utilization (B) and ethanol production (C) rates. Cellobiose and ethanol values represent an average of two independent samples.</p

Homology modeling suggests that a selected mutation in an ABC transporter transmembrane domain (Cphy 2465) in cellulose-adapted populations occurs at a protein-protein interface.

<p>The maltose transporter (3pv0) is shown because its transmembrane domains MalF (green) and MalG (magenta) are the best templates of known structure for Cphy 2465 and Cphy 2464, respectively. A homology model of Cphy 2465 based on MalF places the selected A207V mutation (red) in the coupling helix (arrow and table) that is important in transmitting changes between the transmembrane domains (green and magenta) and the ATPase domains (blue and gold). The mutation occurs in the consensus sequence originally identified in several transporters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086731#pone.0086731-Dassa1" target="_blank">[45]</a>.</p