Timothy R. Filley

Selective adsorption of l- and d-amino acids on calcite: Implications for biochemical homochirality

The emergence of biochemical homochirality was a key step in the origin of life, yet prebiotic mechanisms for chiral separation are not well constrained. Here we demonstrate a geochemically plausible scenario for chiral separation of amino acids by adsorption on mineral surfaces. Crystals of the common rock-forming mineral calcite (CaCO3), when immersed in a racemic aspartic acid solution, display significant adsorption and chiral selectivity of d- and l-enantiomers on pairs of mirror-related crystal-growth surfaces. This selective adsorption is greater on crystals with terraced surface textures, which indicates that d- and l-aspartic acid concentrate along step-like linear growth features. Thus, selective adsorption of linear arrays of d- and l-amino acids on calcite, with subsequent condensation polymerization, represents a plausible geochemical mechanism for the production of homochiral polypeptides on the prebiotic Earth.

Lignin degradation in wood-feeding insects

The aromatic polymer lignin protects plants from most forms of microbial attack. Despite the fact that a significant fraction of all lignocellulose degraded passes through arthropod guts, the fate of lignin in these systems is not known. Using tetramethylammonium hydroxide thermochemolysis, we show lignin degradation by two insect species, the Asian longhorned beetle (Anoplophora glabripennis) and the Pacific dampwood termite (Zootermopsis angusticollis). In both the beetle and termite, significant levels of propyl side-chain oxidation (depolymerization) and demethylation of ring methoxyl groups is detected; for the termite, ring hydroxylation is also observed. In addition, culture-independent fungal gut community analysis of A. glabripennis identified a single species of fungus in the Fusarium solani/Nectria haematococca species complex. This is a soft-rot fungus that may be contributing to wood degradation. These results transform our understanding of lignin degradation by wood-feeding insects.

Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi

Abstract The organic residues produced in the brown-rot (BR) of wood by many basidiomycetes fungi are ubiquitous on most coniferous forest floors. This degraded wood tissue is characterized by low levels of polysaccharides and a high proportion of demethylated lignin with minor glycerol side chain oxidation. Because of the selective enrichment in an aromatic dihydroxy-rich lignin residue, the chemical and biological reactivity of BR degraded wood will be distinctly different from white rot, the other primary class of fungal wood decay, which typically produces oxidized, lignin-depleted residues. The biochemical mechanism by which BR fungi perform this distinctive degradative chemistry is only starting to become known, and molecular studies which examine the chemical changes imparted to lignin over the long-term decay process are lacking. Using 13C-labeled tetramethylammonium hydroxide thermochemolysis (13C-TMAH) and solid state 13C NMR, we investigated the relationship between lignin oxidation/demethylation and polysaccharide metabolism in a 32-week time series study of spruce sapwood inoculated with either of two BR fungi (Postia placenta and Gloeophyllum trabeum). Our findings demonstrate a close relationship between lignin demethylation and polysaccharide loss and suggest demethylation may play a mechanistic role in polysaccharide loss, possibly by assisting in Fenton reactions where catechol/quinone oxidation and cycling aids in iron reduction. The residue remaining after 16 weeks of decay is devoid of polysaccharides, in contrast to the 68% polysaccharide carbon present in the initial spruce, and exhibits an increased aromatic dihydroxy content (resulting from demethylation of the 3-methoxyl carbon) of up to 22% of the lignin, as determined by 13C-TMAH thermochemolysis. In a typical soil or porewater environment these chemical changes would make BR residues highly reactive toward redox sensitive polyvalent metals (e.g. ferric iron) and likely to adsorb to metal hydroxide surfaces.

Tetramethylammonium hydroxide (TMAH) thermochemolysis : proposed mechanisms based upon the application of 13C-labeled TMAH to a synthetic model lignin dimer

The mechanism by which heated tetramethylammonium hydroxide (TMAH) degrades the lignin biopolymer was investigated by the novel application of 13C-labeled TMAH (13C-TMAH) in the thermochemolysis of a synthetic model guaiacyl lignin dimer. GC-MS analysis of the products showed labeling patterns consistent with a base-catalyzed intramolecular displacement of the β-phenoxy group and the formation of two intermediate guaiacyl propane epoxides, a γ-hydroxy-α,β-epoxide and an α-hydroxy-β,γ-epoxide. Methoxide then functions as a nucleophile to open the epoxide ring. These results substantiate the base-catalyzed reactions previously postulated by Gierer (1970) to explain alkali wood pulping and also explain the facile formation and distribution of lignin derivatives obtained in the TMAH thermochemolysis of natural samples. The absence of substantial numbers of bonds involving propyl-aryl ether linkages with adjacent hydroxyl groups is the limiting factor in the complete decomposition of lignin by TMAH thermochemolysis, as propyl-aryl ether linkages without adjacent hydroxyl groups cannot react via this mechanism. This helps to explain why materials such as highly degraded lignin residues, with significant side chain alteration and type III kerogens above the rank of lignite, where aliphatic-aryl ether linkages are thought to be insignificant, are reported to give low yields of TMAH thermochemolysis products.

The application of 13C-labeled tetramethylammonium hydroxide (13C-TMAH) thermochemolysis to the study of fungal degradation of wood.

This paper presents the results from an assessment of the application of a new molecular analytical procedure, 13C-TMAH thermochemolysis, to study the chemical modification of lignin by white-rot and brown-rot fungi. This technique differs from other molecular chemolysis procedures (e.g. TMAH thermochemolysis and CuO alkaline oxidation) as it enables one to determine the amount of hydroxylated aromatic components in degraded lignin residues through a selective lignin depolymerization and 13C-labeled methylation reaction. Major differences were observed in the chemical composition and yield of lignin monomers released from a limited sample set of field and laboratory inoculation brown-rot and white-rot degraded residues when analyzed by 13C-TMAH thermochemolysis. The brown-rot residues were characterized by high yields of 3,4-dihydroxy phenyl compounds, presumably due to fungal demethylation of methoxyl groups on guaiacyl lignin, and relatively low yields of aromatic acids that result from microbial side chain oxidation. The white-rot residues were characterized by low yields of demethylated lignin monomers but relatively high yields of monomers exhibiting side chain oxidation. If generally applicable, this distinct chemical functionality has important implications for the chemical reactivity and solubility of degraded wood residues and consequently the cycling of terrestrial carbon in the geosphere. The 13C-TMAH thermochemolysis procedure provides a rapid and sensitive tool for tracking microbial modifications of lignin in terrestrial environments including coastal sediments, forest soils and waters receiving terrestrial organic matter.

An isotopic biogeochemical assessment of shifts in organic matter input to Holocene sediments from Mud Lake, Florida

Abstract A molecular and isotopic study of sediment cores from a sinkhole lake, Mud Lake, Florida, USA, was performed in order to relate documented changes in the regional terrestrial vegetation and water table over the last ∼5500 years to molecular and isotopic proxies for biological sources of organic matter to the lake sediments. Temporal shifts in the source of organic matter to the sediment, as determined by the stable carbon-isotope composition of bulk organic matter and biomarkers, correspond with previously defined regional scale transitions in forest ecosystems ( Quercus to Pinus at ∼5500 14 C yr BP and Taxodium expansion at 2500 14 C yr BP) and coincident increases in the Floridan water table. The δ 13 C values for total sedimentary organic carbon showed a shift from terrestrial and aquatic macrophyte sources (−27.8‰, in sediments dated at ∼5400 14 C yr BP) within the basal peat, to a cyanobacterial-dominated sapropel (−18.1‰) at the surface. A comparison of the δ 13 C values of bulk sediment and biomarkers representative of cyanobacterial and algal input (e.g. 7- and 8-methylheptadecane and the n -alkane C 17 ) indicates that the present shallow lake was fully developed by ∼2400 14 C yr BP. Differences among the δ 13 C values of specific biomarkers derived from vascular plants (C 29 and C 31 n -alkanes and CuO lignin oxidation products) and from cyanobacteria and algae are nearly equivalent in magnitude to the shift recorded in δ 13 C of TOC, indicating their effective use as source proxies in this system.

Ecosystem effects of non-native earthworms in Mid-Atlantic deciduous forests

In many mid-Atlantic forests where both native and non-native earthworms exist, it is the non-native species that are the dominant component of the soil macrofauna. Few earthworm ecology studies, however, focus attention on these forest systems in order to determine the relative ecological roles and potential interactions of the native and non-native earthworms. In a series of field samplings and experimental manipulations we collected data on the effects of earthworms on below-and aboveground ecosystem processes. Earthworm abundance and the ecological processes measured were dynamic in space and time across the range of study sites. Leaf litter decay rates doubled at sites that had abundant non-native earthworms. Earthworms also altered the abundance of soil fungi, the activity of extracellular enzymes, soil respiration, and the growth of tree seedlings but the effects varied among sites depending on differences in land-use history and forest age. Red oak seedling growth was less at sites that had abundant earthworms but tulip poplar and red maple seedlings grew equally well with and without abundant earthworms. These preliminary results suggest that non-native earthworms have significant ecosystem effects, even in forests where native earthworms still occur. Land use history, however, plays an important role in determining what those effects will be, and these effects are likely to be dynamic, depending on the abundance of non-native earthworms.

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.

Biogeochemical controls on reaction of sedimentary organic matter and aqueous sulfides in holocene sediments of Mud Lake, Florida

Abstract The distribution and quantity of organic sulfur and iron sulfur species were determined in the Holocene sediments from Mud Lake, Florida. The sediments of this shallow, sinkhole lake are characterized by high sulfur and organic carbon contents as well as active sulfate reduction. They record a shift from a basal peat (below 2 m) comprised of water lily-dominated organic matter to the present cyanobacterial/algal-dominated lake deposit (upper 1 m). This shift in depositional environment and subsequent organic matter source was accompanied by variation in the amount of reactive iron delivered to the sediments, which in turn influenced the type and extent of organic matter sulfurization. Extractable intramolecular organic sulfur is principally found as C25 highly branched isoprenoid (HBI) thiolanes. Extractable polysulfide-linked lipids, determined by selective chemical cleavage with MeLi/MeI and analyzed as methylthioethers (MTE), are dominated by n-alkanes with sulfur attachments at position 1 and 2, as well as lower amounts of C25 HBI-MTE. The δ13C values and carbon-chain length distribution of both series of n-alkylMTE indicate that they are derived from distinct biological precursors. Among the n-alkylMTE with sulfur attachment at position 1 there are three homologous series: one saturated and two with both cis and trans enethiol isomers. The identification of the enethiol in the sulfur-linked macromolecules indicates that n-alkylaldehydes are precursors lipids. The intervals of high concentration of bulk organic sulfur and sulfurized lipids coincide with the intervals of high mineral sulfur content (acid volatile sulfide and chromium reducible sulfur). We suggest that the main control on the enhanced addition of sulfur to the organic matter in Mud Lake was the increased formation of polysulfides during the reduction of iron hydroxides and the subsequent reaction of those polysulfides with mildly oxidized sedimentary organic matter.

Belowground competition among invading detritivores

The factors regulating soil animal communities are poorly understood. Current theory favors niche complementarity and facilitation over competition as the primary forms of non-trophic interspecific interaction in soil fauna; however, competition has frequently been suggested as an important community-structuring factor in earthworms, ecosystem engineers that influence belowground processes. To date, direct evidence of competition in earthworms is lacking due to the difficulty inherent in identifying a limiting resource for saprophagous animals. In the present study, we offer the first direct evidence of interspecific competition for food in this dominant soil detritivore group by combining field observations with laboratory mesocosm experiments using 13C and 15N double-enriched leaf litter to track consumption patterns. In our experiments, the Asian invasive species Amynthas hilgendorfi was a dominant competitor for leaf litter against two European species currently invading the temperate deciduous forests in North America. This competitive advantage may account for recent invasion success of A. hilgendorfi in forests with established populations of European species, and we hypothesize that specific phenological differences play an important role in determining the outcome of the belowground competition. In contrast, Eisenoides lonnbergi, a common native species in the Eastern United States, occupied a unique trophic position with limited interactions with other species, which may contribute to its persistence in habitats dominated by invasive species. Furthermore, our results supported neither the hypothesis that facilitation occurs between species of different functional groups nor the hypothesis that species in the same group exhibit functional equivalency in C and N translocation in the soil. We propose that species identity is a more powerful approach to understand earthworm invasion and its impacts on belowground processes.