Jonathan S. Schilling

Synergy between pretreatment lignocellulose modifications and saccharification efficiency in two brown rot fungal systems

Brown rot wood-degrading fungi distinctly modify lignocellulose and completely hydrolyze polysaccharides (saccharification), typically without secreting an exo-acting glucanase and without removing lignin. Although each step of this two-step approach evolved within the same organism, it is unknown if the early lignocellulose modifications are made to specifically facilitate their own abbreviated enzyme system or if enhancements are more general. Because commercial pretreatments are typically approached as an isolated step, answering this question has immense implication on bioprocessing. We pretreated spruce and pine blocks with one of two brown rot fungi, Gloeophyllum trabeum or Fomitopsis pinicola. Wood harvested at weeks 1, 2, 4, and 8 showed a progression of weight loss from time zero due to selective carbohydrate removal. Hemicellulose losses progressed faster than cellulose loss. This “pretreated” material was then saccharified with commercially relevant Trichoderma reesei cellulases or with cellulases from the brown rot fungi responsible for degrading the wood to test for synergy. With increased decay, a significant increase in saccharification efficiency was apparent but not limited to same-species enzyme sources. We also calculated total sugar yields, and calculations that compensate for sugars consumed by fungi suggest a shorter residence time for fungal colonization than calculations based solely on saccharification yields.

Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing.

Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.

Improved pretreatment of lignocellulosic biomass using enzymatically-generated peracetic acid.

Release of sugars from lignocellulosic biomass is inefficient because lignin, an aromatic polymer, blocks access of enzymes to the sugar polymers. Pretreatments remove lignin and disrupt its structure, thereby enhancing sugar release. In previous work, enzymatically generated peracetic acid was used to pretreat aspen wood. This pretreatment removed 45% of the lignin and the subsequent saccharification released 97% of the sugars remaining after pretreatment. In this paper, the amount of enzyme needed is reduced tenfold using first, an improved enzyme variant that makes twice as much peracetic acid and second, a two-phase reaction to generate the peracetic acid, which allows enzyme reuse. In addition, the eight pretreatment cycles are reduced to only one by increasing the volume of peracetic acid solution and increasing the temperature to 60 °C and the reaction time to 6h. For the pretreatment step, the weight ratio of peracetic acid to wood determines the amount of lignin removed.

Oxalate regulation by two brown rot fungi decaying oxalate- amended and non-amended wood

Abstract Oxalic acid secretion by brown rot wood-degrading fungi has been proposed to function in pH control and non-enzymatic biodegradation. Although oxalate production in liquid cultures of brown rot fungi commonly correlates with glucose oxidation, excess oxalate accumulation in wood during oxidative decay could impede Fe3+ reduction by fungal-derived chelators and thus inhibit brown rot. In this study, we pre-treated spruce wood with various oxalate concentrations and subjected it to brown rot decay by Fomitopsis pinicola and Meruliporiaincrassata in agar- and soil-block trials. In agar-block microcosms containing wood pre-treated with 0, 1, 10 or 100 mM sodium oxalate, test fungi equalized wood oxalate and pH at week 12 of decay by either increasing or reducing wood oxalate, depending on the pre-treatment. Oxalate reductions in wood were not accompanied by increases in agar oxalate. During soil-block decay of wood pre-treated with 0 or 50 mM oxalate, oxalate and pH regulation were time-dependent and more variable. Wood oxalate levels did not increase with increasing fungal biomass (per ergosterol); however, decreases in oxalate were not explained by enhanced oxalate catabolism activity, Ca2+ import, or translocation of oxalate into the soil. Our results suggest that brown rot fungi may optimize extracellular oxalate during wood decay, and that soil characteristics may influence this dynamic.

Bioindication of atmospheric heavy metal deposition in the Southeastern US using the moss Thuidium delicatulum

Ectohydric mosses are known accumulators of atmospheric heavy metals. Reliable bioindication of atmospheric heavy metals in the Southern Appalachians using moss has been limited by poor species distribution in moss used in analogous studies. In this study, Pb, Cu, Cr, and Ni concentrations were quantified in the tissue of fern moss Thuidium delicatulum in the central Blue Ridge of Virginia. The objectives of the study were to evaluate the suitability of fern moss for moss-monitoring studies in the Southern Appalachians, to compare local terrestrial metal concentrations, and to test the effects of several geographical and environmental variables on deposition. Fern moss was sampled over four mountains in Virginia following the standard protocol of the German mossmonitoring method. Sampling was standardized for monitoring in deciduous forests, and analysis was performed by graphite furnace-atomic absorption spectrophotometry. Overall concentrations of two metals were significantly different depending on the presence of Pinus spp. in the canopy. Positive and negative correlations of heavy metal concentrations with elevation were also observed, suggesting a need for comprehensive sampling at high and low elevations in mountainous areas. A role for similar moss-monitoring is suggested as a complement to current precipitation analysis techniques and as a compendium for landscape-scale metal monitoring projects. The applications of heavy metal bioindication with this particular species throughout the physiographic province of the Blue Ridge and the Appalachians in future heavy metal deposition studies are discussed. r 2002 Elsevier Science Ltd. All rights reserved.

Lignocellulose modifications by brown rot fungi and their effects, as pretreatments, on cellulolysis

Brown rot fungi Gloeophyllum trabeum and Postia placenta were used to degrade aspen, spruce, or corn stover over 16 weeks. Decayed residues were saccharified using commercial cellulases or brown rot fungal extracts, loaded at equal but low endoglucanase titers. Saccharification was then repeated for high-yield samples using full strength commercial cellulases. Overall, brown rot pretreatments enhanced yields up to threefold when using either cellulase preparation. In the best case, aspen degraded 2 weeks by G. trabeum yielded 72% glucose-from-cellulose, a 51% yield relative to original glucan. A follow-up trial with more frequent harvests showed similar patterns and demonstrated interplay between tissue modifications and saccharification. Hemicellulose and vanillic acid (G6) or vanillin (G4) lignin residues were good predictors of saccharification potential, the latter notable given lignins potential active role in brown rot. Results show basic relationships over a brown rot time course and lend targets for controlling an applied bioconversion process.

Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta

Significance Wood-decomposing fungi are key players in the carbon cycle and are models for making energy from lignocellulose, sustainably. Our study focuses on brown rot fungi that selectively remove carbohydrates, leaving most lignin behind. These fungi often decompose wood faster than their lignin-degrading white rot ancestors, despite losses in genes involved in plant cell wall hydrolysis. To explain brown rot, many have implicated reactive oxygen species (ROS) in facilitating hydrolysis, with microenvironmental gradients partitioning ROS from enzymes. By spatially colocalizing gene expression and enzyme activities as Postia placenta colonizes wood, we provide evidence of an oxidative-hydrolytic two-step mechanism controlled by differential expression, not microenvironments, and we highlight 549 genes (∼4% of the genome) that are upregulated during this unique pretreatment. Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.

Metal Accumulation without Enhanced Oxalate Secretion in Wood Degraded by Brown Rot Fungi

ABSTRACT Brown rot fungi were incubated in agar and agar-wood microcosms containing metallic or hydroxide forms of Al, Cu, and Fe. Metal dissolution was associated with elevated oxalate concentrations in agar, but metals translocated into wood did not affect oxalate accumulation, crystal production, or decay rate, demonstrating a substrate-dependent oxalate dynamic.

Signature Wood Modifications Reveal Decomposer Community History

Correlating plant litter decay rates with initial tissue traits (e.g. C, N contents) is common practice, but in woody litter, predictive relationships are often weak. Variability in predicting wood decomposition is partially due to territorial competition among fungal decomposers that, in turn, have a range of nutritional strategies (rot types) and consequences on residues. Given this biotic influence, researchers are increasingly using culture-independent tools in an attempt to link variability more directly to decomposer groups. Our goal was to complement these tools by using certain wood modifications as ‘signatures’ that provide more functional information about decomposer dominance than density loss. Specifically, we used dilute alkali solubility (DAS; higher for brown rot) and lignin:density loss (L:D; higher for white rot) to infer rot type (binary) and fungal nutritional mode (gradient), respectively. We first determined strength of pattern among 29 fungi of known rot type by correlating DAS and L:D with mass loss in birch and pine. Having shown robust relationships for both techniques above a density loss threshold, we then demonstrated and resolved two issues relevant to species consortia and field trials, 1) spatial patchiness creating gravimetric bias (density bias), and 2) brown rot imprints prior or subsequent to white rot replacement (legacy effects). Finally, we field-tested our methods in a New Zealand Pinus radiata plantation in a paired-plot comparison. Overall, results validate these low-cost techniques that measure the collective histories of decomposer dominance in wood. The L:D measure also showed clear potential in classifying ‘rot type’ along a spectrum rather than as a traditional binary type (brown versus white rot), as it places the nutritional strategies of wood-degrading fungi on a scale (L:D=0-5, in this case). These information-rich measures of consequence can provide insight into their biological causes, strengthening the links between traits, structure, and function during wood decomposition.

High-performance liquid chromatographic analysis of soluble and total oxalate in Ca- and Mg-amended liquid cultures of three wood decay fungi

Abstract Two brown-rot wood decay fungi, Fomitopsis pinicola and Meruliporia incrassata, and the white-rot species Phanerochaete chrysosporium were grown for 4 weeks in liquid culture at 0.35, 0.70, 1.05, and 5.00 mM calcium (Ca) and 1.35 and 2.70 mM magnesium (Mg) concentrations. Soluble and total oxalate levels were quantified using a revised ion-exchange HPLC protocol developed specifically for resolving oxalate and other organic acid anions from medium components. Total oxalate concentrations in brown-rot filtrate were not significantly different among treatments; however, soluble oxalate decreased significantly with increasing Ca concentration. Higher Mg concentrations increased soluble oxalate levels only slightly. There was a significant decrease in medium pH at 5.00 mM Ca for all species, as well as an apparent increase in decarboxylation activity in brown-rot fungi. Total and soluble oxalate levels in the white-rot cultures were generally below detection for all treatments. The results show a significant influence of Ca on soluble oxalate concentrations not seen previously in the brown-rot species Postia placenta.