NMR Characterization of C3H and HCT Down-Regulated Alfalfa Lignin

Independent down-regulation of genes encoding p-coumarate 3-hydroxylase (C3H) and hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) has been previously shown to reduce the recalcitrance of alfalfa and thereby improve the release of fermentable sugars during enzymatic hydrolysis. In this study, ball-milled lignins were isolated from wild-type control, C3H, and HCT gene down-regulated alfalfa plants. One- and two-dimensional nuclear magnetic resonance (NMR) techniques were utilized to determine structural changes in the ball-milled alfalfa lignins resulting from this genetic engineering. After C3H and HCT gene down-regulation, significant structural changes had occurred to the alfalfa ball-milled lignins compared to the wild-type control. A substantial increase in p-hydroxyphenyl units was observed in the transgenic alfalfa ball-milled lignins as well as a concomitant decrease in guaiacyl and syringyl units. Two-dimensional ¹³C–¹H heteronuclear single quantum coherence correlation NMR, one-dimensional distortionless enhancement by polarization transfer-135, and ¹³C NMR measurement showed a noteworthy decrease in methoxyl group and β-O-4 linkage contents in these transgenic alfalfa lignins. ¹³C NMR analysis estimated that C3H gene down-regulation reduced the methoxyl content by ~55–58% in the ball-milled lignin, while HCT down-regulation decreased methoxyl content by ~73%. The gene down-regulated C3H and HCT transgenic alfalfa lignin was largely a p-hydroxyphenyl (H) rich type lignin. Compared to the wild-type plant, the C3H and HCT transgenic lines had an increase in relative abundance of phenylcoumaran and resinol in the ball-milled lignins.     

Influence of Molecular Size and Ligninase Pretreatment on Degradation of Lignins by Xanthomonas sp. Strain 99

The purpose of this study was to examine the relationship between the molecular size of lignin in several preparations and extent of degradation (mineralization) by Xanthomonas sp. strain 99. The influence of ligninase pretreatment was also examined. Five synthetic lignins and one ¹⁴C-methylated spruce lignin were used. The extent of mineralization to ¹⁴CO₂ was greatest for the samples containing the most low-molecular-weight material, and the low-molecular-weight portions were preferentially (or perhaps solely) degraded. Pretreatment of the five synthetic lignins with crude ligninase increased their molecular size and decreased their degradability by the xanthomonad. Pretreatment of the methylated spruce lignin with crude ligninase caused both polymerization and depolymerization but resulted in a net decrease in bacterial degradability. Our results suggest that the xanthomonad can degrade lignins only up to a molecular weight of 600 to 1,000.     

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose

Douglas-fir was SO₂-steam pretreated at different severities (190, 200, and 210 °C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and ¹³C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.     

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.     

Ether linkage between phenolic acids and lignin fractions from wheat straw

The bonding of the bound-phenolic acids present in three lignin preparations isolated from wheat straw where determined. p-Coumaric acid was mainly ester-linked whereas 35–75% of the recovered ferulic acid was ether-linked to milled straw lignin or enzyme lignin. Ferulic acid ethers accounted for 1.1% dry wt of alkali extracted lignin and might explain the high solubility of Gramineae lignins in soda. Isolated lignins were associated to hemicelluloses, principally arabinoglucuronoxylans. The possible existence of ferulic acid cross-links between lignin and arabinoglucuronoxylans is discussed.     

Effect of Penicillium chrysogenum on Lignin Transformation

A strain of Penicillium chrysogenum has been isolated from pine forest soils in Tenerife (Canary Islands). This strain was capable of utilizing hydroxylated and nonhydroxylated aromatic compounds, in particular cinnamic acid, as its sole carbon source. In an optimum medium with high levels of nitrogen (25.6 mM) and low levels of glucose (5.5 mM), it was able to decolorize Poly B-411 and to transform kraft, organosolv, and synthetic dehydrogenative polymerisate lignins. After 30 days of incubation, the amount of recovered kraft lignin was reduced to 83.5 and 91.3% of that estimated for uninoculated controls by spectrophotometry and klason lignin, respectively. At the same time, the pattern of molecular mass distribution of the lignin remaining in cultures was changed. The amount of organosolv lignin recovered from cultures was reduced to 90.1 and 94.6% of the initial amount as evaluated by spectrophotometry and klason lignin, respectively. About 6% of total applied radioactivity of O¹⁴CH₃-organosolv lignin was recovered as ¹⁴CO₂ after 30 days of incubation, and 18.5% of radioactivity from insoluble O¹⁴CH₃-organosolv lignin was solubilized. After 26 days of incubation, 2.9% of ¹⁴C-β-dehydrogenative polymerisate and 4.1% of ¹⁴C-ring-dehydrogenative polymerisate evolved as ¹⁴CO₂.     

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.     

Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Characterization of lignin-rich residues remaining after continuous super-critical water hydrolysis of poplar wood (Populus albaglandulosa) for conversion to fermentable sugars

Poplar wood flour (Populous albaglandulosa) was treated with sub- and super-critical water (subcritical: 325, 350 °C; super-critical: 380, 400, 425 °C) for 60 s at 220 ± 10 atm. Hydrochloric acid (0.05% v/v) was added to samples as acidic catalyst. The final products were separated into water soluble fraction and undegraded solids. The yields of undegraded solids were thoroughly dependent on temperature severity and mainly composed of lignin fragments. Average molecular weights of the lignins were between 1500 and 4400 Da, which was only 1/3-1/8-fold of poplar milled wood lignin (13,250 Da). DFRC (Derivatization Followed by Reductive Cleavage) analysis revealed that C6C3 phenols (coniferyl and sinapyl alcohol) were rarely detected in the lignins, indicating occurrence of two probable lignin reactions during SCW hydrolysis: lignin fragmentation via splitting of β-O-4 linkage and loss of propane side chains. These results were also confirmed by ¹H and ¹³C NMR spectroscopic analysis.     

Structural characterization of alkaline hydrogen peroxide pretreated grasses exhibiting diverse lignin phenotypes

Background

For cellulosic biofuels processes, suitable characterization of the lignin remaining within the cell wall and correlation of quantified properties of lignin to cell wall polysaccharide enzymatic deconstruction is underrepresented in the literature. This is particularly true for grasses which represent a number of promising bioenergy feedstocks where quantification of grass lignins is particularly problematic due to the high fraction of p-hydroxycinnamates. The main focus of this work is to use grasses with a diverse range of lignin properties, and applying multiple lignin characterization platforms, attempt to correlate the differences in these lignin properties to the susceptibility to alkaline hydrogen peroxide (AHP) pretreatment and subsequent enzymatic deconstruction.

Results

We were able to determine that the enzymatic hydrolysis of cellulose to to glucose (i.e. digestibility) of four grasses with relatively diverse lignin phenotypes could be correlated to total lignin content and the content of p-hydroxycinnamates, while S/G ratios did not appear to contribute to the enzymatic digestibility or delignification. The lignins of the brown midrib corn stovers tested were significantly more condensed than a typical commercial corn stover and a significant finding was that pretreatment with alkaline hydrogen peroxide increases the fraction of lignins involved in condensed linkages from 88?C95% to ~99% for all the corn stovers tested, which is much more than has been reported in the literature for other pretreatments. This indicates significant scission of ??-O-4 bonds by pretreatment and/or induction of lignin condensation reactions. The S/G ratios in grasses determined by analytical pyrolysis are significantly lower than values obtained using either thioacidolysis or 2DHSQC NMR due to presumed interference by ferulates.

Conclusions

It was found that grass cell wall polysaccharide hydrolysis by cellulolytic enzymes for grasses exhibiting a diversity of lignin structures and compositions could be linked to quantifiable changes in the composition of the cell wall and properties of the lignin including apparent content of the p-hydroxycinnamates while the limitations of S/G estimation in grasses is highlighted.     

Carbon-13 nuclear magnetic resonance spectra of lignins

From the ¹³C-nmr spectra of a large number of dimeric and monomeric lignin model compounds the chemical shifts of the carbon atoms of the C₉-units in lignin with different substitution patterns were determined. The absorption peaks of the carbon-13 spectra of two lignins (beech and spruce) could be assigned by comparison (Table 3).     

The properties of syringyl, guaiacyl and p-hydroxyphenyl artificial lignins

1. Artificial lignins have been produced on potato parenchyma. 2. The methoxyl-free lignin and 4-hydroxy-3-methoxy (guaiacyl) lignins could be estimated by the sulphuric acid method but the 4-hydroxy-3,5-dimethoxy (syringyl) lignins could not. 3. Permanganate oxidation of isolated p-coumaric lignin gave 4-hydroxybenzoic acid, 4-hydroxyisophthalic acid and small amounts of hydroxytrimesic acid and 4-hydroxyphthalic acid. Ferulic lignin gave vanillic acid and 5-carboxyvanillic acid and also small amounts of 4-hydroxybenzoic acid and dehydrodivanillic acid. The sinapic lignin gave traces of syringic acid and of 4-hydroxybenzoic acid. 4. The p-coumaric lignin is a highly condensed polymer. The ferulic lignin is partly uncondensed and partly condensed through the 5-position like gymnosperm lignin. The sinapic lignin shows no evidence of condensation and is probably an ether-linked polymer.     

Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Enzymatic demethylation of lignin for potential biobased polymer applications

Lignin is a highly methylated, recalcitrant biopolymer available aplenty in nature, and is highly heteropolymer in nature, but yet it has been an under-utilized biopolymer. Modifying it chemically, biologically or enzymatically could render it a good candidate for phenol formaldehyde resin or into fine chemicals, fuels, and plastics applications. Lignin demethylation is facilitated by the enzymes called the O-demethylases, which are able to strip-off of the –OCH₃ group in lignin, that give rise to the more widely accessible phenolic hydroxyls groups. Biological demethylation of lignins can be accomplished by means of the microorganisms, such as the white-rot, soft-rot and brown-rot fungi, besides some species of bacteria. Although the enzymes responsible for the lignin demethylation process have not been identified and purified adequately, it is perhaps possible that the O-demethylases, which have the ability to remove the O-methyl groups at the C-3 and (or) C-4 positions of the benzyl ring of low molecular weight lignin-like model compounds (LMCs) and lignin makes them the suitable candidate. These LMCs resemble the aromatic moieties inherent in the molecular structure of lignins, such as the vanillate, syringate, and veratrate. Thus, these enzymes are known as vanillate-O-demethylases, syringate O-demethylases, veratrate O-demethylases and Tetrahydrofolate (THF)-dependent O-demethylase (LigM), respectively. Whereas, some ligninolytic enzymes are known to cause damage to the structure of lignins (e.g., laccases, manganese-dependent peroxidase and lignin peroxidases). The O-demethylase enzymes are believed to be capable of removing the O-methyl groups from the lignins without affecting the complex backbone structure of the lignins. The mechanism of action of O-demethylases on lignin degradation is still largely unexplored, and their ability to remove the O-methyl groups from lignins has not been elucidated sufficiently. In this review, the recent advances made on the molecular approaches in the lignin demethylation (O-demethylases and ligninolytic enzymes), degradation and the probable strategies to tone up the lignin quality have been discussed in detail. The demethylation process of lignins by means of enzymes is envisaged to open up new vistas for its application as a biopolymer in various bioprocess and biorefinery process.     

Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium

Phanerochaete chrysosporium degraded purified Kraft lignin, alkali-extracted and dioxane-extracted straw lignin, and lignosulfonates at a similar rate, producing small-molecular-weight (~1,000) soluble products which comprised 25 to 35% of the original lignins. At concentrations of 1 g of lignin liter⁻¹, 90 to 100% of the acid-insoluble Kraft, alkali straw, and dioxane straw lignins were degraded by 1 g of fungal mycelium liter⁻¹ within an active ligninolytic period of 2 to 3 days. Cultures with biomass concentrations as low as 0.16 g liter⁻¹ could also completely degrade 1 g of lignin liter⁻¹ during an active period of 6 to 8 days. The absorbance at 280 nm of 2 g of lignosulfonate liter⁻¹ increased during the first 3 days of incubation and decreased to 35% of the original value during the next 7 days. The capacity of 1 g of cells to degrade alkali-extracted straw lignin under optimized conditions was estimated to be as high as 1.0 g day⁻¹. This degradation occurred with a simultaneous glucose consumption rate of 1.0 g day⁻¹. When glucose or cellular energy resources were depleted, lignin degradation ceased. The ability of P. chrysosporium to degrade the various lignins in a similar manner and at very low biomass concentrations indicates that the enzymes responsible for lignin degradation are nonspecific.     

Short communication: Isolation of a bacterium capable of limited degradation of industrial and labelled,natural and synthetic lignins

Pseudomonas putida, isolated from decomposing plant materials, degraded several lignin-related aromatic compounds. After 30 days of incubation in media containing polymeric Kraft-lignin (PKL), the amount of Klason lignin had decreased by about 13%. When ¹⁴C-labelled dehydropolymers of coniferyl alcohol (DHP) lignins and ¹⁴C-lignin-lignocelluloses were used as substrates, mineralization to ¹⁴CO₂ by the P. putida strain ranged from 1.4% to 2.1%.     

Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic ¹⁴C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the ¹⁴CO₂ evolved. Average percentages of the [ring-¹⁴C]- and [side chain-¹⁴C]-lignins, respectively, recovered as ¹⁴CO₂ at the cessation of ¹⁴CO₂ evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.     

Effect of hydrolytic lignin on formation of protein–lignin complexes and protein degradation by rumen microbes

Several methods have been developed to protect feed protein from rumen microbial degradation. The current study aimed to evaluate the potential use of an industrial lignin, namely hydrolytic lignin, to protect protein from rumen microbial degradation. The hydrolytic lignins assessed in this study were extracted from wheat straw previously subjected to various steam treatment conditions (pressure: 15, 17 and 19 bar; reaction time: 0, 5 and 10 min; use of acidic catalyst: without and with 2% H₂SO₄ on DM basis). Results indicated that hydrolytic lignin can precipitate protein when measured by a standard bovine serum albumin assay. It was also observed that protein-precipitating capacity of lignin increased with increasing harshness of steam treatment until a point from which no further effect was observed. The effect of lignin upon protein degradation in vitro was clearly detected. Both ammonia nitrogen and iso-acid concentration in vitro were significantly decreased (P<0.01) when lignin was added to fermentation flask containing casein. Unlike tannins, hydrolytic lignins do not inhibit rumen microbial activity. Additionally, it was observed that lignin’s ability to bind and protect protein is a pH-dependent reaction. Protein binding to lignin is markedly reduced at pH<3.0.     

Lignocellulose Mineralization by Arctic Lake Sediments in Response to Nutrient Manipulation

Mineralization of specifically labeled ¹⁴C-cellulose- and ¹⁴C-lignin-labeled lignocelluloses by Toolik Lake, Alaska, sediments was examined in response to manipulation of various environmental factors. Mineralization was measured by quantifying the amount of labeled CO₂ released from the specifically labeled substrates. Nitrogen (NH₄NO₃) and, to a greater degree, phosphorus (PO₄⁻³) additions enhanced the mineralization of white pine (Pinus strobus) cellulose during the summer of 1978. Nitrogen and phosphorus together had no cumulative effect. During the summer of 1979, nitrogen or phosphorus alone had only a slight stimulatory effect on the mineralization of a sedge (Carex aquatilis) cellulose; however, together, they had a dramatic effect. This variable response of mineralization to nutrient addition between 1978 and 1979 was probably attributable to year-to-year variation in nutrient availability within the lake. Cellobiose addition and oxygen depletion inhibited the amount of pine cellulose mineralized. Whereas addition of nitrogen to oxygen-depleted treatments had limited effect, addition of phosphorus resulted in mineralizations equal to or greater than that of the controls. Nitrogen had no effect on mineralization of pine or Carex lignins. Phosphorus, however, inhibited mineralization of both lignins. With Carex lignin, the phosphorus inhibition occurred at a concentration as low as 0.1 μM. The antagonistic role of phosphorus in cellulose and lignin mineralizations may be of significance in understanding the increased proportion of lignin relative to cellulose in decomposing litter.