Molecular underpinnings of methyl jasmonate-induced resistance in Norway spruce.

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm.

Styrene, (+)-trans-(1R,4S,5S)-4-Thujanol and Oxygenated Monoterpenes Related to Host Stress Elicit Strong Electrophysiological Responses in the Bark Beetle Ips typographus.

Bark beetles kill apparently vigorous conifers during epidemics by means of pheromone-mediated aggregation. During non-endemic conditions the beetles are limited to use trees with poor defense, like wind-thrown. To find olfactory cues that help beetles to distinguish between trees with strong or weak defense, we collected volatiles from the bark surface of healthy felled or standing Picea abies trees. Furthermore, living trees were treated with methyl jasmonate in order to induce defense responses. Volatiles were analyzed by combined gas chromatography and electroantennographic detection (GC-EAD) on Ips typographus antennae. Compounds eliciting antennal responses were characterized by single sensillum recording for identification of specific olfactory sensory neurons (OSN). Release of monoterpene hydrocarbons decreased, while oxygenated compounds increased, from spring to early summer in felled trees. In both beetle sexes particular strong EAD activity was elicited by trace amounts of terpene alcohols and ketones. 4-Thujanol gave a very strong response and the absolute configuration of the tested natural product was assigned to be (+)-trans-(1R,4S,5S)-thujanol by stereoselective synthesis and enantioselective gas chromatography. One type of OSN responded to all ketones and five other OSN were characterized by the type of compounds that elicited responses. Three new OSN classes were found. Of the eight EAD-active compounds found in methyl jasmonate-treated bark, the known anti-attractant 1,8-cineole was the one most strongly induced. Our data support the hypothesis that highly active oxygenated host volatiles could serve as positive or negative cues for host selection in I. typographus and in other bark beetles.

CO2 enrichment alters diurnal stem radius fluctuations of 36-yr-old Larix decidua growing at the alpine tree line.

To understand how trees at high elevations might use water differently in the future, we investigated the effects of CO2 enrichment and soil warming (separately and combined) on the water relations of Larix decidua growing at the tree line in the Swiss Alps. We assessed diurnal stem radius fluctuations using point dendrometers and applied a hydraulic plant model using microclimate and soil water potential data as inputs. Trees exposed to CO2 enrichment for 9 yr showed smaller diurnal stem radius contractions (by 46 ± 16%) and expansions (42 ± 16%) compared with trees exposed to ambient CO2 . Additionally, there was a delay in the timing of daily maximum (40 ± 12 min) and minimum (63 ± 14 min) radius values for trees growing under elevated CO2 . Parameters optimized with the hydraulic model suggested that CO2 -enriched trees had an increased flow resistance between the xylem and bark, representing a more buffered water supply system. Soil warming did not alter diurnal fluctuation dynamics or the CO2 response. Elevated CO2 altered the hydraulic water flow and storage system within L. decidua trees, which might have contributed to enhanced growth during 9 yr of CO2 enrichment and could ultimately influence the future competitive ability of this key tree-line species.

A transcriptomic network underlies microstructural and physiological responses to cadmium in Populus x canescens.

Bark tissue of Populus × canescens can hyperaccumulate cadmium, but microstructural, transcriptomic, and physiological response mechanisms are poorly understood. Histochemical assays, transmission electron microscopic observations, energy-dispersive x-ray microanalysis, and transcriptomic and physiological analyses have been performed to enhance our understanding of cadmium accumulation and detoxification in P. × canescens. Cadmium was allocated to the phloem of the bark, and subcellular cadmium compartmentalization occurred mainly in vacuoles of phloem cells. Transcripts involved in microstructural alteration, changes in nutrition and primary metabolism, and stimulation of stress responses showed significantly differential expression in the bark of P. × canescens exposed to cadmium. About 48% of the differentially regulated transcripts formed a coregulation network in which 43 hub genes played a central role both in cross talk among distinct biological processes and in coordinating the transcriptomic regulation in the bark of P. × canescens in response to cadmium. The cadmium transcriptome in the bark of P. × canescens was mirrored by physiological readouts. Cadmium accumulation led to decreased total nitrogen, phosphorus, and calcium and increased sulfur in the bark. Cadmium inhibited photosynthesis, resulting in decreased carbohydrate levels. Cadmium induced oxidative stress and antioxidants, including free proline, soluble phenolics, ascorbate, and thiol compounds. These results suggest that orchestrated microstructural, transcriptomic, and physiological regulation may sustain cadmium hyperaccumulation in P. × canescens bark and provide new insights into engineering woody plants for phytoremediation.

Could rapid diameter changes be facilitated by a variable hydraulic conductance?

Adequate radial water transport between elastic bark tissue and xylem is crucial in trees, because it smoothens abrupt changes in xylem water potential, greatly reducing the likelihood of suffering dangerous levels of embolism. The radial hydraulic conductance involved is generally thought to be constant. Evidence collected about variable root and leaf hydraulic conductance led us to speculate that radial hydraulic conductance in stem/branches might also be variable and possibly modulated by putative aquaporins. We therefore correlated diameter changes in walnut (Juglans regia L.) with changes in water potential, altered by perfusion of twig samples with D-mannitol solutions having different osmotic potentials. Temperature and cycloheximide (CHX; a protein synthesis inhibitor) treatments were performed. The temperature response and diameter change inhibition found in CHX-treated twigs underpinned our hypothesis that radial hydraulic conductance is variable and likely mediated by a putative aquaporin abundance and/or activity. Our data demonstrate that radial water transport in stem/branches can take two routes in parallel: an apoplastic and a cell-to-cell route. The contribution of either route depends on the hydraulic demand and is closely linked to a boost of putative aquaporins, causing radial conductance to be variable. This variability should be considered when interpreting and modelling diameter changes.

Hot water and dilute acid pretreatment of high and low specific gravity Populus deltoides clones.

Populus sp. are hardwood feedstocks that grow in forest management areas that are logged for softwoods; however, they are also being considered as an energy-destined feedstock. The objective of this work was to determine the effect of xylose yield from dilute acid and hot water pretreatments performed in unstirred batch stainless steel reactors at temperatures ranging from 140 to 200°C. Populus deltoides clones S13C20 and S7C15 used in this study originated from Eastern Texas and were cultivated for 14 years in Pine Tree, AR. P. deltoides clones S13C20 and S7C15 had specific gravities of 0.48 and 0.40, respectively. Bark and wood were examined separately. As expected, hot water pretreatments, in the tested temperature range, resulted in very little direct xylose recovery. However, the 140°C dilute acid pretreatment of the lower specific gravity clone, S7C15, wood yielded the highest average xylose recovery of 56%. This condition also yielded the highest concentration of furfural, 9 mg/g sample, which can be inhibitory to the fermentation step. The highest xylose recovery from bark samples, 31%, was obtained with clone S7C15, using the 160°C dilute acid pretreatment for 60 min.

Gene discovery for the bark beetle-vectored fungal tree pathogen Grosmannia clavigera.

BACKGROUND: Grosmannia clavigera is a bark beetle-vectored fungal pathogen of pines that causes wood discoloration and may kill trees by disrupting nutrient and water transport. Trees respond to attacks from beetles and associated fungi by releasing terpenoid and phenolic defense compounds. It is unclear which genes are important for G. clavigera's ability to overcome antifungal pine terpenoids and phenolics. RESULTS: We constructed seven cDNA libraries from eight G. clavigera isolates grown under various culture conditions, and Sanger sequenced the 5' and 3' ends of 25,000 cDNA clones, resulting in 44,288 high quality ESTs. The assembled dataset of unique transcripts (unigenes) consists of 6,265 contigs and 2,459 singletons that mapped to 6,467 locations on the G. clavigera reference genome, representing ~70% of the predicted G. clavigera genes. Although only 54% of the unigenes matched characterized proteins at the NCBI database, this dataset extensively covers major metabolic pathways, cellular processes, and genes necessary for response to environmental stimuli and genetic information processing. Furthermore, we identified genes expressed in spores prior to germination, and genes involved in response to treatment with lodgepole pine phloem extract (LPPE). CONCLUSIONS: We provide a comprehensively annotated EST dataset for G. clavigera that represents a rich resource for gene characterization in this and other ophiostomatoid fungi. Genes expressed in response to LPPE treatment are indicative of fungal oxidative stress response. We identified two clusters of potentially functionally related genes responsive to LPPE treatment. Furthermore, we report a simple method for identifying contig misassemblies in de novo assembled EST collections caused by gene overlap on the genome.

Abietane and C20-norabietane diterpenes from the stem bark of Fraxinus sieboldiana and their biological activities.

Fourteen new abietane (1-14) and seven new C(20)-norabietane (15-21) diterpenes, together with five known analogues, have been isolated from the stem bark of Fraxinus sieboldiana. Their structures were elucidated by spectroscopic data analysis. In the in vitro assays, at 10(-5) M, compounds 8, 16, and 22 showed inhibitory activity against the release of ß-glucuronidase in rat polymorphonuclear leukocytes induced by platelet-activating factor, with 59.7 ± 4.8%, 56.1 ± 5.6%, and 65.9 ± 3.1% inhibition, respectively. Compound 23 was active against H5N1 avian influenza virus with an IC(50) value of 4.8 µM. Compounds 3 and 5 exhibited selective cytotoxic activities against A2780 (IC(50) 1.7 µM) and A549 (IC(50) 6.0 µM), respectively.

Gene expression pattern in response to wounding, methyl jasmonate and ethylene in the bark of Hevea brasiliensis.

Natural rubber production in Hevea brasiliensis is determined by both tapping and ethephon frequencies. It is affected by a complex physiological disorder called tapping panel dryness. This syndrome is likely to be induced by environmental and latex harvesting stresses. Defence responses, including rubber biosynthesis, are dramatically mediated by wounding, jasmonate and ethylene (ET), among other factors. Using real-time RT-PCR, the effects of wounding, methyl jasmonate (MeJA) and ET on the relative transcript abundance of a set of 25 genes involved in their signalling and metabolic pathways were studied in the bark of 3-month-old epicormic shoots. Temporal regulation was found for 9 out of 25 genes. Wounding treatment regulated the transcript abundance of 10 genes. Wounding-specific regulation was noted for the HbMAPK, HbBTF3b, HbCAS1, HbLTPP and HbPLD genes. MeJA treatment regulated the transcript abundance of nine genes. Of these, the HbMYB, HbCAS2, HbCIPK and HbChi genes were shown to be specifically MeJA inducible. ET response was accompanied by regulation of the transcript abundance of eight genes, and six genes, HbETR2, HbEIN2, HbEIN3, HbCaM, HbPIP1 and HbQM, were specifically regulated by ET treatment. Additionally, the transcript level of the HbGP and HbACR genes was enhanced by all three treatments simultaneously. Overall, a large number of genes were found to be regulated 4 h after the treatments were applied. This study nevertheless revealed some jasmonic acid-independent wound signalling pathways in H. brasiliensis, provided a general characterization of signalling pathways and will serve as a new base from which to launch advanced studies of the network of pathways operating in H. brasiliensis.

Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production.

BACKGROUND AND AIMS: The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. METHODS: Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. KEY RESULTS: Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. CONCLUSION: The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.

Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae).

Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10microg/ml for UD, 6microM for celecoxib and 42microM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.

Growth and wood/bark properties of Abies faxoniana seedlings as affected by elevated CO2.

Growth and wood and bark properties of Abies faxoniana seedlings after one year's exposure to elevated CO2 concentration (ambient + 350 (+/- 25) micromol/mol) under two planting densities (28 or 84 plants/m(2)) were investigated in closed-top chambers. Tree height, stem diameter and cross-sectional area, and total biomass were enhanced under elevated CO2 concentration, and reduced under high planting density. Most traits of stem bark were improved under elevated CO2 concentration and reduced under high planting density. Stem wood production was significantly increased in volume under elevated CO2 concentration under both densities, and the stem wood density decreased under elevated CO2 concentration and increased under high planting density. These results suggest that the response of stem wood and bark to elevated CO2 concentration is density dependent. This may be of great importance in a future CO2 enriched world in natural forests where plant density varies considerably. The results also show that the bark/wood ratio in diameter, stem cross-sectional area and dry weight are not proportionally affected by elevated CO2 concentration under the two contrasting planting densities. This indicates that the response magnitude of stem bark and stem wood to elevated CO2 concentration are different but their response directions are the same.

Methyl jasmonate treatment of mature Norway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protects against infection by Ceratocystis polonica, a bark beetle-associated fungus.

When conifers such as Picea abies Karst. (Norway spruce) are attacked by insects or pathogens, they often respond by producing increased quantities of terpenoid oleoresin. This response can be mimicked in young P. abies seedlings by treatment with methyl jasmonate (MJ). In this study, we determined the effects of MJ on terpenoids and other chemical defenses of mature P. abies, and investigated whether this treatment protected trees against attack by the blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau, the most important fungal associate of the spruce bark beetle Ips typographus L. Methyl jasmonate treatment induced the formation of traumatic resin ducts in the developing xylem, enhanced resin flow and stimulated increased accumulation of monoterpenes, sesquiterpenes and diterpene resin acids. However, only minor changes were detected in terpene composition in response to MJ treatment and no changes in soluble phenolic concentration were measured. There was much variability in the timing and degree of response to MJ among clones. The observed chemical and anatomical changes in response to MJ treatment were correlated with increased resistance to C. polonica, suggesting that terpenoid oleoresin may function in defense against this pathogen.

Uptake of phenol from aqueous solutions by adsorption in a Pinus pinaster bark packed bed.

The adsorption of phenol from aqueous solutions using a column packed with pre-treated Pinus pinaster bark was studied. The influence of the inlet phenol concentration (0.01 or 0.1 g/L) and the flow rate (6, 15.6 or 30 mL/min) on the breakthrough curves was analysed. An increase in the flow rate, decreased the time necessary to reach the breakthrough point and, for the highest inlet concentration, the dynamic capacity of the bed, from 7.5 to 0.4 min and from 0.38 to 0.15 mg phenol/go.d. bark, respectively, at 0.1 g/L. The LUB Design Approach was used to determine the equivalent length of unused bed. The lower LUB values, which imply a better utilization of the bark bed, were obtained at the higher flow rate. A model which considered the effect of axial dispersion was successfully used to describe the fixed-bed operation behaviour for the lower flow rates. For the lowest inlet phenol concentration, the axial dispersion coefficient increased significantly when the flow rate increased.

Identification of differentially expressed genes and signalling pathways in bark of Hevea brasiliensis seedlings associated with secondary laticifer differentiation using gene expression microarray.

The natural rubber of Para rubber tree, Hevea brasiliensis, is the main crop involved in industrial rubber production due to its superior quality. The Hevea bark is commercially exploited to obtain latex, which is produced from the articulated secondary laticifer. The laticifer is well defined in the aspect of morphology; however, only some genes associated with its development have been reported. We successfully induced secondary laticifer in the jasmonic acid (JA)-treated and linolenic acid (LA)-treated Hevea bark but secondary laticifer is not observed in the ethephon (ET)-treated and untreated Hevea bark. In this study, we analysed 27,195 gene models using NimbleGen microarrays based on the Hevea draft genome. 491 filtered differentially expressed (FDE) transcripts that are common to both JA- and LA-treated bark samples but not ET-treated bark samples were identified. In the Eukaryotic Orthologous Group (KOG) analysis, 491 FDE transcripts belong to different functional categories that reflect the diverse processes and pathways involved in laticifer differentiation. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and KOG analysis, the profile of the FDE transcripts suggest that JA- and LA-treated bark samples have a sufficient molecular basis for secondary laticifer differentiation, especially regarding secondary metabolites metabolism. FDE genes in this category are from the cytochrome (CYP) P450 family, ATP-binding cassette (ABC) transporter family, short-chain dehydrogenase/reductase (SDR) family, or cinnamyl alcohol dehydrogenase (CAD) family. The data includes many genes involved in cell division, cell wall synthesis, and cell differentiation. The most abundant transcript in FDE list was SDR65C, reflecting its importance in laticifer differentiation. Using the Basic Local Alignment Search Tool (BLAST) as part of annotation and functional prediction, several characterised as well as uncharacterized transcription factors and genes were found in the dataset. Hence, the further characterization of these genes is necessary to unveil their role in laticifer differentiation. This study provides a platform for the further characterization and identification of the key genes involved in secondary laticifer differentiation.

Genome-wide identification and expression analysis of the metacaspase gene family in Hevea brasiliensis.

Metacaspases, a family of cysteine proteases, have been suggested to play important roles in programmed cell death (PCD) during plant development and stress responses. To date, no systematic characterization of this gene family has been reported in rubber tree (Hevea brasiliensis). In the present study, nine metacaspase genes, designated as HbMC1 to HbMC9, were identified from whole-genome sequence of rubber tree. Multiple sequence alignment and phylogenetic analyses suggested that these genes were divided into two types: type I (HbMC1-HBMC7) and type II (HbMC8 and HbMC9). Gene structure analysis demonstrated that type I and type II HbMCs separately contained four and two introns, indicating the conserved exon-intron organization of HbMCs. Quantitative real-time PCR analysis revealed that HbMCs showed distinct expression patterns in different tissues, suggesting the functional diversity of HbMCs in various tissues during development. Most of the HbMCs were regulated by drought, cold, and salt stress, implying their possible functions in regulating abiotic stress-induced cell death. Of the nine HbMCs, HbMC1, HbMC2, HbMC5, and HbMC8 displayed a significantly higher relative transcript accumulation in barks of tapping panel dryness (TPD) trees compared with healthy trees. In addition, the four genes were up-regulated by ethephon (ET) and methyl jasmonate (MeJA), indicating their potential involvement in TPD resulting from ET- or JA-induced PCD. In summary, this work provides valuable information for further functional characterization of HbMC genes in rubber tree.

Methyl jasmonate induces changes mimicking anatomical defenses in diverse members of the Pinaceae.

Conifers have defenses such as the production of phenolic compounds and resins that can be induced by bark beetles and other invading organisms, but the signaling agents involved are unknown. The anatomical effects of methyl jasmonate (MJ), a potent inducer of certain plant defenses, were compared with wounding of the bark of 12-15-year-old trees of five conifer species. Wounding in all species resulted in tissue necrosis and wound periderm development immediately around the wound site. One cm from the wound, swelling of phloem polyphenolic parenchyma cells and phenolic accumulation were observed in Pseudotsuga menziesii (Mirb.) Franco, Picea pungens Engelman, Larix occidentalis Nutt. and Pinus monticola Douglas ex D. Don, but not in Taxus brevifolia Nutt. Traumatic resin ducts were formed in response to wounding in three species of Pinaceae, but not in P. monticola, which formed irregular clusters of cells rather than ducts. Taxus brevifolia did not form resin ducts in response to either wounding or MJ treatment. In the Pinaceae species studied, surface application of 100 mM MJ caused similar anatomical changes to those observed in response to wounding, including phenolic accumulation, cell swelling and traumatic resin duct formation, but it did not induce a wound periderm. Traumatic resin ducts differed in size among the study species, ranging from small in L. occidentalis to very large in P. menziesii. In P. menziesii, P. pungens and L. occidentalis, traumatic resin ducts were more abundant after MJ treatment than after wounding. We conclude that the octadecanoid pathway is likely involved in defense responses in stems of the Pinaceae, but not necessarily in other taxa.

Enzymatic degradation of Elephant grass (Pennisetum purpureum) stems: influence of the pith and bark in the total hydrolysis.

The internal pith of a high energy plant, Elephant grass (EG), was more extensively degraded (>50% dry matter) compared to the outer cortex (31%) or the whole stem (35%) by an enzyme preparation from Humicola insolens, Ultraflo. Reducing sugars and acetic acid release from the pith was also higher compared to the cortex. Supplementation of Ultraflo with a type-C feruloyl esterase increased the level of deacetylation but also led to reduced solubilisation. The addition of 20% dimethyl sulfoxide (DMSO) as a co-solvent also reduced the solubility of EG by Ultraflo, although acetic acid release was increased, complimenting previous results found on model substrates. The presence of DMSO was also shown to have a protective effect on xylanase activity but not acetyl esterase activity in Ultraflo. Xylan in the biomass was preferentially solubilised by DMSO, while Ultraflo removed more glucose than xylose.

The effect of bark on sulfur dioxide-ethanol-water fractionation and enzymatic hydrolysis of forest biomass.

The focus of this study was to find out the effect of bark on SO2-ethanol-water (SEW) fractionation and enzymatic hydrolysis of forest biomass. Softwood bark was found to be more harmful than hardwood bark in both processes. For softwood, the amount of undigested wood in SEW fractionation increased with the increasing bark content, whereas the hardwood bark did not impair the fractionation of wood. The higher the softwood bark content was the lower were the yields in enzymatic hydrolysis likely due to the unproductive binding of enzymes on lignin and other compounds. Addition of surfactant Tween 20 (2% w/w on substrate) prior to enzyme more than doubled the sugar yield of bark-rich softwood pulp. Hardwood bark impaired enzymatic hydrolysis when its share was over 28%. According to a preliminary study, lignosulfonates from the carry-over liquor seem to improve the sugar yield in the enzymatic hydrolysis by acting as a surfactant.

Impact of pretreatments on morphology and enzymatic saccharification of shedding bark of Melaleuca leucadendron.

The effects of subcritical water (SCW) and dilute acid pretreatments on the shedding bark of Melaleuca leucadendron (paper bark tree, PBT) biomass morphology, crystallinity index (CrI) and enzymatic saccharification were studied. The morphology of PBT bark was characterized by X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. SCW pretreatment mainly extracted amorphous parts of the biomass hence its CrI increased, partial decrystallization of cellulose and exposing of intact nanofibers of cellulose were observed for SCW pretreatment at 180°C. On the other hand, dilute acid pretreatment at 160°C exhibited a large decrease in CrI, an increase in surface area, a decrease in lignin content and decrystallization of cellulose as well as the peel-off and degradation of some nanofiber bundles. Dilute acid and SCW pretreatments of PBT biomass resulted in about 4.5 fold enhancement in glucose release relative to the untreated one.