Microbial Delignification with White Rot Fungi Improves Forage Digestibility

Three wild-type white rot fungi and two cellulase-less mutants developed from Phanerochaete chrysosporium K-3 (formerly Sporotrichum pulverulentum) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy. P. chrysosporium K-3 and Ceriporiopsis subvermispora FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However, C. subvermispora removed the most lignin and significantly improved biodegradation over all other treatments. Phellinus pini RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from P. chrysosporium K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that C. subvermispora extensively removed ester-linked p-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that C. subvermispora removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues). C. subvermispora improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.     

Changes in Phenolics Distribution After Chemical Pretreatment and Enzymatic Conversion of Miscanthus × giganteus Internode

In addition to lignin, grass cell walls are characterized by the presence of hydroxycinnamic acids that play a significant role in cross-linking polymers into a cohesive network, and pretreatments are required to overcome the recalcitrance of lignocelluloses prior to enzymatic bioconversion of polysaccharides. The effects of dilute acid and ammonium hydroxide pretreatments were studied on the chemical composition and enzymatic saccharification of Miscanthus internodes fragments. The hydroxycinnamic acid content was reduced after both pretreatments, while lignin got enriched in condensed linked structures. In addition, dilute acid pretreatment was effective in decreasing xylan content of Miscanthus, while ammonia treatment induced a marked swelling effect on the cell walls of parenchyma, vascular sclerenchyma, and epidermal sclerenchyma. The phenol distribution at the cell level was estimated using UV transmission microspectrophotometry. Internode cell walls displayed different UV spectra according to the cell type. However, the secondary cell walls had similar UV spectra after pretreatment, whereas spectra recorded at the cell corner region displayed variations according to cell type and pretreatment. Acid pretreatment was more efficient than ammonia to improve the conversion of polysaccharides by a Trichoderma cellulolytic cocktail. Although pretreatments achieved moderate saccharification yields, the secondary cell walls were altered at some pit regions of the vascular sclerenchyma whereas parenchyma appeared recalcitrant. Variations in the UV spectra of enzyme-digested cell walls suggest pretreatment-dependent heterogeneity of the phenolic distribution in the more recalcitrant cell walls.     

Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background  

Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions.     

Creative biological lignin conversion routes toward lignin valorization

Lignin, the largest renewable aromatic resource, is a promising alternative feedstock for the sustainable production of various chemicals, fuels, and materials. Despite this potential, lignin is characterized by heterogeneous and macromolecular structures that must be addressed. In this review, we present biological lignin conversion routes (BLCRs) that offer opportunities for overcoming these challenges, making lignin valorization feasible. Funneling heterogeneous aromatics via a ‘biological funnel’ offers a high-specificity bioconversion route for aromatic platform chemicals. The inherent aromaticity of lignin drives atom-economic functionalization routes toward aromatic natural product generation. By harnessing the ligninolytic capacities of specific microbial systems, powerful aromatic ring-opening routes can be developed to generate various value-added products. Thus, BLCRs hold the promise to make lignin valorization feasible and enable a lignocellulose-based bioeconomy.     

New and classic families of secreted fungal heme peroxidases

Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the “classic” fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.     

Anaerobic Biodegradation of the Lignin and Polysaccharide Components of Lignocellulose and Synthetic Lignin by Sediment Microflora

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses and [¹⁴C-polysaccharide]lignocelluloses were prepared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush, and a hardwood. These [¹⁴C]lignocellulose preparations and synthetic [¹⁴C]lignin were incubated anaerobically with anoxic sediments collected from a salt marsh, a freshwater marsh, and a mangrove swamp. During long-term incubations lasting up to 300 days, the lignin and polysaccharide components of the lignocelluloses were slowly degraded anaerobically to ¹⁴CO₂ and ¹⁴CH₄. Lignocelluloses derived from herbaceous plants were degraded more rapidly than lignocellulose derived from the hardwood. After 294 days, 16.9% of the lignin component and 30.0% of the polysaccharide component of lignocellulose derived from the grass used (Spartina alterniflora) were degraded to gaseous end products. In contrast, after 246 days, only 1.5% of the lignin component and 4.1% of the polysaccharide component of lignocellulose derived from the hardwood used (Rhizophora mangle) were degraded to gaseous end products. Synthetic [¹⁴C]lignin was degraded anaerobically faster than the lignin component of the hardwood lignocellulose; after 276 days, 3.7% of the synthetic lignin was degraded to gaseous end products. Contrary to previous reports, these results demonstrate that lignin and lignified plant tissues are biodegradable in the absence of oxygen. Although lignocelluloses are recalcitrant to anaerobic biodegradation, rates of degradation measured in aquatic sediments are significant and have important implications for the biospheric cycling of carbon from these abundant biopolymers.     

Production of useful modified lignin polymers by bioconversion of lignocellulose with Streptomyces

Lignin degrading strains of Streptomyces were grown on lignocelluloses from a variety of plant sources. These actinomycetes readily degraded the lignin present in the residues and released a major portion of the lignin into the growth medium as a water soluble, modified polymer. The polymer, an acid precipitable polyphenolic lignin (APPL), was recovered from spent culture media by acid precipitation or dialysis/lyophilization. APPL's were shown to be mostly free of nonlignin components. As compared to native lignin they were more oxidized, were especially enriched in phenolic hydroxyl groups, and were significantly reduced in methoxyl groups. The yield of APPL from different lignocelluloses correlated with their biodegradability. Grasses such as corn stover were the optimal lignocellulose type for APPL production by Streptomyces. In contrast white-rot fungi produced only small amounts of APPL as they decomposed lignin. A solid state bioconversion process was developed using Streptomyces viridosporus T7A to produce APPL from corn stover lignocellulose in yields >or= 30% of the initial lignin present in the substrate. APPL produced by S. viridosporus was examined for its properties and possible use as an antioxidant. The APPL was shown to have good antioxidant properties after mild chemical treatment to reduce the alpha-carbonyl groups present in the APPL. Oxidation of the APPL with hydroxyl radical (OH(*)) further improved its antioxidant properties probably as the result of aromatic ring hydroxylation reactions. As compared with currently used commercial antioxidants, the modified APPL was thought to be competitive when economics of production was considered. Native lignin on the other hand was shown to exhibit no antioxidant properties, even after reduction and/or oxidation.     

Biotechnological production of vanillin

Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.     

Preferential degradation of phenolic lignin units by two white rot fungi.

The differential biodegradation of phenolic and nonphenolic (C-4-etherified) lignin units in wheat straw treated with the white rot fungi Pleurotus eryngii and Phanerochaete chrysosporium was investigated under solid-state fermentation conditions. Two analytical techniques applied to permethylated straw were used for this purpose, i.e., alkaline CuO degradation and analytical pyrolysis (both followed by gas chromatography-mass spectrometry for product identification). Despite differences in the enzymatic machinery produced, both ligninolytic fungi caused a significant decrease in the relative amount of phenolic lignin units during the degradation process. Nevertheless, no differences in the biodegradation rates of phenolic and etherified cinnamic acids were observed. Changes in lignin composition and cinnamic acid content were also analyzed in the phenolic and nonphenolic lignin moieties. The results obtained are discussed in the context of the enzymatic mechanisms of lignin biodegradation.     

Degradation of Softwood, Hardwood, and Grass Lignocelluloses by Two Streptomyces Strains

Two Streptomyces strains, S. viridosporus T7A and S. setonii 75Vi2, were grown on softwood, hardwood, and grass lignocelluloses, and lignocellulose decomposition was followed by monitoring substrate weight loss, lignin loss, and carbohydrate loss over time. Results showed that both Streptomyces strains substantially degraded both the lignin and the carbohydrate components of each lignocellulose; however, these actinomycetes were more efficient decomposers of grass lignocelluloses than of hardwood or softwood lignocelluloses. In particular, these Streptomyces strains were more efficient decomposers of grass lignins than of hardwood or softwood lignins.     

Bioconversion of Phenolic Monomers of Lignin and Lignin-Containing Substrates by the Basidiomycete <Emphasis Type="Italic">Lentinus tigrinus</Emphasis>

The bioconversion of phenolic monomers of lignin (veratrol, vanillin, and vanillyl alcohol), hydrolyzed lignin, and sodium lignosulfonate (a product of the chemical modification of native lignin) by the basidiomycete Lentinus tigrinus was studied. It was found that the growth of the fungi on lignin monomer compounds is suppressed. A noticeable growth of the fungal biomass was observed only on the technical substrate sodium lignosulfonate. A comprehensive physicochemical study of the products of microbial transformation of sodium lignosulfonate was performed. It was established that the main direction of lignin bioconversion is oxidative condensation to form humic substances. In this case, depolymerization of the phenolic skeleton of lignin to monomeric phenol derivatives did not occur. The aromatic carbon atoms of the phenolic skeleton, unlike the carbon atoms of polysaccharides, were not involved in the fungal biomass growth. The observed growth of the fungus on the technical substrate sodium lignosulfonate can be explained by the presence of admixtures of oligomeric polysaccharides hemicellulose and cellulose, which can be used by the fungus as a carbon source.     

Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis

In the near future, grasses must provide most of the biomass for the production of renewable fuels. However, grass cell walls are characterized by a large quantity of hydroxycinnamic acids such as ferulic and p‐coumaric acids, which are thought to reduce the biomass saccharification. Ferulic acid (FA) binds to lignin, polysaccharides and structural proteins of grass cell walls cross‐linking these components. A controlled reduction of FA level or of FA cross‐linkages in plants of industrial interest can improve the production of cellulosic ethanol. Here, we review the biosynthesis and roles of FA in cell wall architecture and in grass biomass recalcitrance to enzyme hydrolysis.     

Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches

Biodegradation can achieve complete and cost-effective elimination of aromatic pollutants through harnessing diverse microbial metabolic processes. Aromatics biodegradation plays an important role in environmental cleanup and has been extensively studied since the inception of biodegradation. These studies, however, are diverse and scattered; there is an imperative need to consolidate, summarize, and review the current status of aromatics biodegradation. The first part of this review briefly discusses the catabolic mechanisms and describes the current status of aromatics biodegradation. Emphasis is placed on monocyclic, polycyclic, and chlorinated aromatic hydrocarbons because they are the most prevalent aromatic contaminants in the environment. Among monocyclic aromatic hydrocarbons, benzene, toluene, ethylbenzene, and xylene; phenylacetic acid; and structurally related aromatic compounds are highlighted. In addition, biofilms and their applications in biodegradation of aromatic compounds are briefly discussed. In recent years, various biomolecular approaches have been applied to design and understand microorganisms for enhanced biodegradation. In the second part of this review, biomolecular approaches, their applications in aromatics biodegradation, and associated biosafety issues are discussed. Particular attention is given to the applications of metabolic engineering, protein engineering, and “omics” technologies in aromatics biodegradation.     

Influence of nutritional supplementation on solid-substrate fermentation of wheat straw with an alkaliphilic white-rot fungus (Coprinus sp.)

Summary The solid-substrate fermentation of wheat straw with an alkaliphilic white-rot fungus (Coprinus sp.) was found to be influenced by the levels of nitrogen, phosphorus+sulphur and free carbohydrates, in terms of biodegradation of straw ingredients, microbial protein production and changes in in-vitro dry matter digestibility (IVDMD). Nitrogen and Phosphorus+Sulphur compounds favoured the bioconversion and their optimum levels were (g/100 g DM): urea (sterile): 1.5, urea (unsterile): 3.0; superphosphate: 1.0. The addition of free carbohydrates as molasses and whey had detrimental effect on biodegradation of lignin as also on organic matter degradation and digestibility. However, the protein production was enhanced in the supplemented straw. The optimized laboratory fermentation was also extended to 4 kg-(sterile and unsterile) and 50 kg-(unsterile) fermentation.     

Pretreatment of poplar lignocellulose by gamma-ray or ozone for subsequent fungal biodegradation

Summary Poplar lignocelluloses, ¹⁴C-labelled, on all the cell wall components or only the lignin moieties, were either irradiated with -rays from ⁶⁰Co or treated with ozone. The two pretreatments increase the accessibility of cellulose to commercial cellulase and enhance, to the same extent, lignin and polysaccharide biodegradation by Phanerochaete chrysosporium. As far as delignification is concerned ozone treatment appears, however, to be the most efficient through its effects both on lignin solubilization and lignin biodegradation. Ozone treatment and fungal biodegradation, of poplar sawdust increase its in vitro digestibility when performed independently. Moreover, we have shown that when these treatments are sequentially associated, they make the digestibility of sawdust comparable to that of straw, provided that the pH of the culture medium is controlled. These results open possibilities for the use of such transformed raw materials as animal feed.     

Evaluation of CuO oxidation parameters for determining the source and stage of lignin degradation in soil

The composition of phenols and other aromatic compounds in organic and mineral soil horizons and their respective source vegetation from different climatic zones of the Canadian Prairies were analyzed using CuO oxidation and gas chromatography-mass spectrometry (GC-MS) to investigate the stage of lignin degradation. Parameters based on the CuO oxidation products were calculated for the soils and corresponding vegetation to determine the lignin sources and to monitor the lignin degradation. In addition to the widely used lignin monomer parameters, parameters resulting from lignin-derived phenolic dimers are used for the first time to assess lignin degradation in soils. The composition of lignin-derived phenols (S/V, C/V) in soil closely matches the composition observed in their respective source plants (grass, Aspen, Pine) reflecting the preservation of characteristic lignin patterns in soils. Degradation parameters based on lignin phenols and benzenes derived from tannins or other phenolic biomolecules indicate a progressive degradation from the vegetation to the soil horizons. In addition to commonly used lignin monomer indicators, parameters based on the lignin dimers are applied. Lignin degradation is found to be lowest in the Pine forest, intermediate in the grassland soils and highest in the Aspen-grassland transition soil. Degradation parameters based on non-lignin aromatic derivatives (3,5-dihydroxybenzoic acid, benzenepolycarboxylic acids) demonstrate a similar trend. The lignin from samples in the cooler climate (Black Chernozems) is observed to be more oxidized than in the soils from the warmer climate (Brown Chernozems) suggesting that abiotic processes may be in involved in the alteration of lignin and other phenolic biomolecules in soils. The results indicate that the comparative analysis of CuO oxidation products of soils and source vegetation is a valuable tool to assess the sources and degradation of lignin in soils.     

Changes in the composition of hydrolyzed lignin during composting

Microbial assemblages were composed for composting hydrolyzed lignin. Data on bioconversion of aromatic compounds with various types of substitution in die ring were used for this purpose. Composting of hydrolyzed lignin reduced the contents of lignin, low-hydrolyzable polysaccharides, resins, and low-molecularweight phenols and resulted in accumulation of humic acids. The resulting compost showed no phytotoxicity.     

Degradation of extractive-free lignocelluloses by Coriolus versicolor and Poria placenta

Summary The wood-decay fungi Coriolus versicolor, a white-rot fungus, and Poria placenta, a brown-rot fungus, were grown on an extractive-free lignocellulose prepared from quackgrass (Agropyron repens). Their abilities to decompose this lignocellulose were compared to their abilities to decompose softwood (Picea pungens) and hardwood (Acer rubrum) lignocelluloses. The two fungi were grown on malt-extract dampened lignocelluloses at 28°C for up to 12 weeks. Replicate cultures were periodically harvested and lignocellulose decomposition was followed by monitoring substrate weight loss, lignin loss, and carbohydrate loss. Coriolus versicolor decomposed the lignin and carbohydrate components of the grass lignocellulose as efficiently as the softwood and hardwood lignocelluloses. Poria placenta, however, was not an efficient degrader of either lignin or carbohydrate in the grass lignocellulose. Poria placenta readily decomposed carbohydrate components of the softwood lignocellulose but not the hardwood lignocellulose.Paper number 81520 of the Idaho Agricultural Experiment Station