Tailoring microbes to upgrade lignin

Lignin depolymerization generates a mixture of numerous compounds that are difficult to separate cost-effectively. To address this heterogeneity issue, microbes have been employed to ‘biologically funnel’ a broad range of compounds present in depolymerized lignin into common central metabolites that can be converted into a single desirable product. Because the composition of depolymerized lignin varies significantly with the type of biomass and the depolymerization method, microbes should be selected and engineered by considering this compositional variation. An ideal microbe must efficiently metabolize all relevant lignin-derived compounds regardless of the compositional variation of feedstocks, but discovering or developing such a perfect microbe is very challenging. Instead, developing multiple tailored microbes to tolerate a given mixture of lignin-derived compounds and to convert most of these into a target product is more practical. This review summarizes recent progress toward the development of such microbes for lignin valorization and offers future directions.     

Leaf litter decomposition in a southern Sonoran Desert ecosystem, northwestern Mexico: Effects of habitat and litter quality

Leaf litter decomposition of dominant woody perennial species in the three most common habitats of the southern Sonoran Desert was studied using the litter-bag method. Our objective was to assess the influence of litter quality on decomposition rates in three contrasting desert environments. The hypotheses were: (1) decomposition rates within the same litter type are faster in more mesic habitats, (2) decomposition rates are lower in higher lignin content or lower nutrient quality substrates, and (3) species-rich substrates enhance decomposition rates. For all litter types and habitats, a rapid loss of mass occurred during the summer rains at the start of the experiment, but total loss within the same litter type differed significantly among habitats. Decay rates were not higher in the more mesic habitat, but in the dry plains where solar irradiance and termite activity were highest. While termite activity was less important in the arroyos and absent in the hillsides habitats, proliferation of fungal mycelium in these sites was much higher than in the plains, suggesting that biotic and abiotic factors act both independently of litter richness. Lignin content seems to be an important factor controlling the loss of litter, because decay rates were inversely related to litter initial lignin content in all three habitats. Leaf litter diversity did not enhance rates of decomposition. The leaf litter mixture had k-values similar to the most recalcitrant monospecific litter in all three habitats, indicating a neutral or even antagonistic role of species-specific compounds in decomposition rates.     

Mild hydrogenolysis of in-situ and isolated Pinus radiata lignins

The Pd/C-catalysed hydrogenolysis of in-situ and isolated lignins from Pinus radiata wood was investigated to gain a more complete understanding of the factors affecting yield and composition of the hydrogenolysis products. Such hydrogenolysis products could potentially be refined into aromatic feedstock chemicals providing sustainable alternatives to petroleum-derived phenols. Lignins were converted into solvent-soluble oils composed of monomeric, dimeric and oligomeric products in high yields, up to 89% of the original lignin. The main monomer products were dihydroconiferyl alcohol and 4-n-propyl guaiacol. Dimeric and oligomeric compounds constituted 75% of the hydrogenolysis oils and were mainly composed of dihydroconiferyl alcohol and 4-n-propyl guaiacol units linked by β-5, 5-5, 4-O-5 and β-1 linkages. Hydrogenolysis of steam exploded wood gave lower yields of lignin hydrogenolysis products compared to unmodified wood due to fewer reactive aryl-ether linkages in the lignin.     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Ligninolysis

Lignin, the most abundant renewable source of aromatic carbon on earth, consists in a highly irregular three dimensional biopolymer of oxygenated phenylpropanoid units. In natural environments, lignin is only degraded efficiently by some basidioamycetes that secrete an array of enzymes for this purpose. Recent advances in our understanding of the mechanism of lignin breakdown have revealed several features that make this process highly unique from a biochemical standpoint. This article summarizes some of them.     

Micromechanics and anatomical changes during early ontogeny of two lianescent Aristolochia species

 The mechanical properties of young stems of Aristolochia macrophylla Lam. and Aristolochia brasiliensis Mart. et Zucc. were studied during elongation growth and primary differentiation. Data for the modulus of elasticity, for the viscoelastic behaviour caused by longitudinal tension and for the shear modulus resulting from torsion around a longitudinal axis were related to the underlying structural changes by quantitative analysis of stem anatomy, tissue distribution, ultrastructure, and cell wall biochemistry. The orientation of cellulose microfibrils was determined by light microscopy and small-angle X-ray diffraction, and the lignin content was determined by thioglycolic acid derivatization and spectroscopic quantification. It was demonstrated that the increase in stability during early development is due to the complementary effects of increase in cell wall material, lignification, and cellulose microfibril alignment. A detailed micromechanical model, considering internal prestresses, is proposed to explain the characteristic biphasic stress-strain behaviour as well as the strain-hardening observed. Received: 22 March 1999 / Accepted 9 September 1999     

Screening of Polish Fir Honeydew Honey Using GC/MS,HPLC‐DAD,and Physical‐Chemical Parameters: Benzene Derivatives and Terpenes as Chemical Markers

GC/MS of headspace solid phase micro extraction (HS‐SPME) and solvent extractives along with targeted HPLC‐DAD of Polish fir (Abies alba Mill .) honeydew honey (FHH), were used to determine the chemical profiles and potential markers of botanical origin. Additionally, typical physical‐chemical parameters were also assigned. The values determined for FHH were: conductivity (1.2 mS/cm), water content (16.7 g/100 g), pH (4.5), and CIE chromaticity coordinates (L* = 48.4, a* = 20.6, b* = 69.7, C* = 72.9, and h° = 73.5). FHH contained moderate‐high total phenolic content (533.2 mg GAE/kg) and antioxidant activity (1.1 mmol TEAC/kg) and (3.2 mmol Fe²⁺/kg) in DPPH and FRAP assays. The chemical profiles were dominated by source plant‐originated benzene derivatives: 3,4‐dihydroxybenzoic acid (up to 8.7 mg/kg, HPLC/honey solution), methyl syringate (up to 14.5%, GC/solvent extracts) or benzaldehyde (up to 43.7%, GC/headspace). Other markers were terpenes including norisoprenoid (4‐hydroxy‐3,5,6‐trimethyl‐4‐(3‐oxobut‐1‐enyl)cyclohex‐2‐en‐1‐one, up to 20.3%, GC/solvent extracts) and monoterpenes, mainly linalool derivatives (up to 49%, GC/headspace) as well as borneol (up to 5.9%, GC/headspace). The application of various techniques allowed comprehensive characterisation of FHH. 4‐Hydroxy‐3,5,6‐trimethyl‐4‐(3‐oxobut‐1‐enyl)cyclohex‐2‐en‐1‐one, coniferyl alcohol, borneol, and benzaldehyde were first time proposed for FHH screening. Protocatechuic acid may be a potential marker of FFH regardless of the geographical origin.     

Use of diluted medium in repeated-batch fermentation for production of lignin peroxidase by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Lignin peroxidase has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by the white rot fungus, Phanerochaete chrysosporium, include the improvement of yield results and cell maintenance. In the present work, Phanerochaete chrysosporium was immobilized in polyurethane foam and used for repeated-batch fermentations with various dilution of the initial medium (D), and lignin peroxidase production was investigated. The peak of 283 ± 17.5 U lignin peroxidase/l production rate was obtained at a D of 1/5, with significantly lower production rates seen at higher and lower dilution ratios. When six cycles of repeated-batch fermentation were conducted using a D of 1/5, the results revealed that at least four cycles of repeated-batch fermentation were possible with a high lignin peroxidase production rate under a cut-off value of 178 ± 3.87 U/l. Furthermore, the cell-free culture broth could be successfully concentrated to 2,800 U/l by ultrafiltration. Thus, the present study shows that optimizing the dilution of the utilized nutritional medium can improve repeated batch production of lignin peroxidase from immobilized P. chrysosporium, in terms of both cycle number and output.