Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

Production of microbial protein from tree bark by Phanerochaete chrysosporium

Phanerochaete chrysosporium was grown in fermentors on NaOH-extracted maple, pine, and cedar barks at the optimum substrate concentration of 1% (w/v). The yields (mg protein/liter) on maple, pine, and cedar were 1500, 1200, and 880, respectively, which are probably due to the different lignin contents of the barks. Lignin is not utilized. The productivities at 30°C obtained for pine (4.07 × 10^?2 g protein/liter hr) and cedar (2.63 × 10^?2 g protein/liter hr) barks were greater than for maple (2.63 × 10^?2 g protein/liter hr). The substrate (bark) was the limiting component of the fermentation. Over the 26–38°C temperature range protein productivity increased by a factor of three (1.55 × 10^?2 vs. 4.61 × 10^?2 g protein/liter hr) for maple bark. Low agitation rates resulted in an overproduction of cellulase and reduced levels of microbial protein.     

Changes to the N cycle following bark beetle outbreaks in two contrasting conifer forest types

Outbreaks of Dendroctonus beetles are causing extensive mortality in conifer forests throughout North America. However, nitrogen (N) cycling impacts among forest types are not well known. We quantified beetle-induced changes in forest structure, soil temperature, and N cycling in Douglas-fir (Pseudotsuga menziesii) forests of Greater Yellowstone (WY, USA), and compared them to published lodgepole pine (Pinus contorta var. latifolia) data. Five undisturbed stands were compared to five beetle-killed stands (4–5 years post-outbreak). We hypothesized greater N cycling responses in Douglas-fir due to higher overall N stocks. Undisturbed Douglas-fir stands had greater litter N pools, soil N, and net N mineralization than lodgepole pine. Several responses to disturbance were similar between forest types, including a pulse of N-enriched litter, doubling of soil N availability, 30–50 % increase in understory cover, and 20 % increase in foliar N concentration of unattacked trees. However, the response of some ecosystem properties notably varied by host forest type. Soil temperature was unaffected in Douglas-fir, but lowered in lodgepole pine. Fresh foliar %N was uncorrelated with net N mineralization in Douglas-fir, but positively correlated in lodgepole pine. Though soil ammonium and nitrate, net N mineralization, and net nitrification all doubled, they remained low in both forest types (<6 μg N g soil^?1 NH₄ ⁺or NO₃ ^?; <25 μg N g soil^?1 year^?1 net N mineralization; <8 μg N g soil^?1 year^?1 net nitrification). Results suggest that beetle disturbance affected litter and soil N cycling similarly in each forest type, despite substantial differences in pre-disturbance biogeochemistry. In contrast, soil temperature and soil N–foliar N linkages differed between host forest types. This result suggests that disturbance type may be a better predictor of litter and soil N responses than forest type due to similar disturbance mechanisms and disturbance legacies across both host–beetle systems.     

Alkali solubility of hemicelluloses in relation to delignification

The polysaccharide composition of bark from Pinus radiata, Salix fragilis, and Populus euramericana has been determined. All the barks contained lower levels of cellulose and hemicellulose than the corresponding woods; cellulose: hemicellulose ratios were also lower in the barks. Alkali extracted all of the hemicellulose-A but only half of the hemicellulose-B from P. radiata bark without prior delignification. Similar alkaline extraction removed almost all of the hemicellulose (A + B) from ryegrass leaves without delignification. With the other samples tested only a part of the hemicellulose A and B is extracted without delignification. It is suggested that the polysaccharide so extracted represents wall hemicellulose which is not linked to lignin or other wall constituents by alkali-stable links.     

Effects of Two-Stage Dilute Acid Pretreatment on the Structure and Composition of Lignin and Cellulose in Loblolly Pine

A standard two-step dilute sulfuric acid pretreatment was performed on Loblolly pine to enhance the overall efficiency of enzymatic deconstruction of woody biomass to monomeric sugars. The structure of milled wood lignin and cellulose isolated from the untreated and acid-treated biomass was studied in detail. Solid-state ¹³C NMR spectroscopy coupled with line shape analyses has been employed to elucidate cellulose crystallinity and ultrastructure. The results indicate an increase in the degree of crystallinity and reduced relative proportion of less ordered cellulose allomorphs following the acid pretreatment. This increase was attributed to a preferential degradation of amorphous cellulose and less ordered crystalline forms during the high temperature pretreatment. Milled wood lignin structural elucidation by quantitative ¹³C and ³¹P NMR reveals an increase in the degree of condensation of lignin due to the pretreatment. The increase in degree of condensation is accompanied by a decrease in β-O-4 linkages which were fragmented and recondensed during the high temperature acid-catalyzed reactions.     

Solid-State NMR Investigation of Bio-chars Produced from Biomass Components and Whole Biomasses

Bio-char is a by-product from thermochemical treatment of biomass and has been identified as an energy condensed product with a comparable heating value as commercial coal. However, the combustion of such solid product as an energy resource is only a preliminary application. It is highly possible to convert bio-char, which always has a condensed aromatic and porous structure to various high-value products. The investigations of the structures and formation pathways for the bio-char are very important to any future applications. In this study, six different biomass components, including cellulose, lignin, and tannin, and three whole biomasses—pine wood, pine residue, and pine bark—have been used to produce bio-char at 400, 500, and 600 °C. Solid-state NMR and FT-IR have been employed in this study to characterize the structures for the bio-chars. The results indicated that the bio-chars produced from lignin contained some methoxyl groups, and the bio-chars produced from tannin contained significantly higher amount of phenolic hydroxyl groups. Compared to the bio-chars produced from pine wood and residue, the bio-chars produced from pine bark contained more aromatic C–O bonds, and aliphatic C–O and C–C bonds, which may be due to the significantly higher amount of lignin and tannin in the pine bark. However, the elevated amounts of aromatic C–O and aliphatic C–O and C–C bonds in the bio-chars from pine bark appeared to be completely decomposed at 600 °C.     

Fuel Characteristics of Solid Biofuel Derived from Oil Palm Biomass and Fast Growing Timber Species in Malaysia

The fuel characteristics of solid biofuels derived from biomass that is abundantly available in Malaysia are presented. The objectives of the study were to characterize fuel properties of oil palm biomass (empty fruit bunch (EFB) and oil palm trunk (OPT)) and wood from a range of fast growing timber species (Albizia falcataria, Acacia spp., Endospermum spp. and Macaranga spp.), inclusive and exclusive of bark. Among the fast-growing timber species, the higher heating values ranged from 4288 cal g^-1 to 4383 cal g^-1 for wood inclusive of bark, and 4134 cal g^-1 to 4343 cal g^-1 for wood exclusive of bark. The inclusive of bark portion in the biomass sample generally increased the heating value except for Macaranga spp. Empty fruit bunch and oil palm trunk had heating values of 4315 cal g^-1 and 4104 cal g^-1, respectively. Ash-forming elements and trace elements were much higher in the timber species samples inclusive of bark than samples exclusive of bark. On the other hand, oil palm biomass contained higher ash-forming elements and trace elements than the wood from the fast growing timber species. The European energy crops show higher HHV, Cl and Si content but lower K, Mg, Na and P compared to the local biomass used in this study. The data obtained from this study can serve as a foundation for the selection of suitable biomass to be used as solid fuel, or as a reference on the fabrication of conversion systems for the selection of biomass solid fuel.     

Influence of temperature,pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K_m and V_max determined for substrate guaiacol were 15.4 μM and 372.9 IU mg^?1, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg^?1. Moreover, the metal ions, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺ and Cd²⁺, especially Hg²⁺, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.     

Differentiating manool and 13-epimanool with NMR chiral shift reagents

Use of NMR chiral shift reagents showed that a compound isolated from Pinus contorta bark and Tsuga heterophylla wood was 13-epimanool. Southern pine (Pinus spp.) tall oil contained a mixture of manool and 13-epimanool.     

Isolation and Structural Characterization of Lignin Polymer from Dendrocalamus sinicus

Dendrocalamus sinicus, which is the largest bamboo species in the world, has broad prospects in the fields of bioenergy and biorefinery application. In this study, dewaxed D. sinicus samples were sequentially treated with 80 % ethanol containing 0.025 M HCl, 80 % ethanol containing 0.5 % NaOH, and aqueous alkaline solutions (containing 2.0, 5.0, and 8.0 % NaOH, respectively) at 75 °C for 4 h, in which 9.63, 8.71, 21.83, 21.09, and 13.09 % of the original lignin were isolated, respectively. The lignin fractions obtained were comparatively characterized by chemical composition, molecular weights, and structural features by wet chemical and instrumental analysis methods. It was found that the bamboo lignin fractions isolated by ethanol had lower weight-average molecular weights (1,360–1,380 g?mol^?1) and contained much higher amounts of associated hemicelluloses, while the lignin fractions isolated by aqueous alkaline solutions had higher weight-average molecular weights (5,300–6,040 g?mol^?1) and contained lower amounts of associated hemicelluloses. Spectroscopy analyses indicated that the bamboo lignin was a typical grass lignin, consisting of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. A small percentage of the lignin side-chain was found to be acetylated at the γ-carbon, predominantly at syringyl units. The major interunit linkages present in the bamboo lignin obtained were β-O-4′ aryl ether linkages, together with lower amounts of β-β′, β-5′, and β-1′ linkages.     

Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study

A rapid and mild extraction protocol for the preparation of lignin was achieved by microwave-assisted heating in formic acid at 101 °C under atmospheric pressure. In this case, birch lignin was extracted with microwave heating process (ML) in formic acid and characterized by elemental analysis, FTIR, GPC, ¹H NMR and ¹³C–¹H HSQC. In addition, the antioxidant activity of the samples was investigated. For comparative study, milled wood lignin (MWL) and lignin extracted with oil bath heating process (OL) were prepared. The results showed that the lignin yield under microwave heating was much higher than that under oil bath heating. A maximal delignification degree (89.77%) was achieved when microwave heating time was 30 min. When double time (60 min) was used under oil bath heating, the delignification degree was 66.11%. The structural characterization showed that the lignin structure of ML did not change dramatically, which is a mixture of GS-type with β-O-4′ ether bond as the major inter-unit linkage. As for antioxidant activity against DPPH, the radical scavenging index (RSI) of ML was 1.20, which was higher than that of MWL (0.53), suggesting that ML exhibited much higher antioxidant activity than MWL.     

Isolation of a Bacterium Capable of Degrading Peanut Hull Lignin

Thirty-seven bacterial strains capable of degrading peanut hull lignin were isolated by using four types of lignin preparations and hot-water-extracted peanut hulls. One of the isolates, tentatively identified as Arthrobacter sp., was capable of utilizing all four lignin preparations as well as extracted peanut hulls as a sole source of carbon. The bacterium was also capable of degrading specifically labeled [¹⁴C]lignin-labeled lignocellulose and [¹⁴C]cellulose-labeled lignocellulose from the cordgrass Spartina alterniflora and could also degrade [¹⁴C]Kraft lignin from slash pine. After 10 days of incubation with [¹⁴C]cellulose-labeled lignocellulose or [¹⁴C]lignin-labeled lignocellulose from S. alterniflora, the bacterium mineralized 6.5% of the polysaccharide component and 2.9% of the lignin component.     

A Decade of Streamwater Nitrogen and Forest Dynamics after a Mountain Pine Beetle Outbreak at the Fraser Experimental Forest,Colorado

Forests of western North America are currently experiencing extensive tree mortality from a variety of bark beetle species, and insect outbreaks are projected to increase under warmer, drier climates. Unlike the abrupt biogeochemical changes typical after wildfire and timber harvesting, the outcomes of insect outbreaks are poorly understood. The mountain pine bark beetle (Dendroctonus ponderosae) began to attack lodgepole pine (Pinus contorta) at the Fraser Experimental Forest in 2002 and spread throughout the research area by 2007. We compared streamwater nitrogen (N) from 2003 through 2012 with data from the previous two decades in four watersheds with distinct forest management histories, stand structures, and responses to the beetle outbreak. Watersheds dominated by old-growth had larger trees and lost 85% of overstory pine and 44% of total basal area to bark beetles. In contrast, managed watersheds containing a mixture of second-growth (30–60 year old) and old-growth (250- to 350-year old) had higher density of subcanopy trees, smaller mean tree diameter, and lower bark beetle-induced mortality (~26% of total basal area). Streamwater nitrate concentrations were significantly higher in old-growth watersheds during the outbreak than pre-outbreak levels during snowmelt and base flow seasons. In mixed-age stands, streamwater nitrate concentrations were unaffected by the outbreak. Beetle outbreak elevated inorganic N export 43 and 74% in two old-growth watersheds though the amounts of N released in streamwater were low (0.04 and 0.15 kg N ha^?1) relative to atmospheric inputs (<2% of annual N deposition). Increased height, diameter, and foliar N of measured in residual live trees augmented demand for N, far in excess of the change in N export during the outbreak. Reallocation of soil resources released after pine mortality to overstory and understory vegetation helps explain high nutrient retention in watersheds affected by bark beetle outbreaks.     

Production of compost from marine waste: evaluation of the product for use in ecological agriculture

The sea contains large amounts of resources that are sometimes considered as waste. Such material includes the waste generated by the fish-processing industry and seaweed that is washed up on shores. In this study, these waste products were windrow composted, along with pine bark as a source of carbon and aeration. The final mix proportions were 20 % seaweed, 20 % fish waste, and 60 % pine bark (v/v). After 10 weeks, stable, well-structured, hygienic compost, which was rich in organic matter and nutrients and had a low metal content, was obtained. Tests for maturity, hygiene, and phytotoxicity, along with a detailed physical and chemical characterization, showed that this compost can be used as an organic amendment and/or growth substrate for use in ecological agriculture. The only limiting feature was the high salinity, which could easily be lowered prior to composting the material.     

Chemical Changes During 6 Years of Decomposition of 11 Litters in Some Canadian Forest Sites. Part 2. 13C Abundance, Solid-State 13C NMR Spectroscopy and the Meaning of “Lignin”

There is still a poor understanding of how changes in the organic composition of litter contribute to slowing or even cessation of decomposition. Using ¹³C nuclear magnetic resonance (NMR) spectroscopy of samples from the Canadian Intersite Decomposition Experiment (CIDET), we asked whether increasing lignin per se could account for the well-known increase in acid-unhydrolyzable residue (AUR), and secondly, using three litters from four sites with different mean annual temperatures, whether changes in organic composition would follow similar trajectories with C mass loss. At 6 years, there was 16–39% C remaining for 10 foliar litters and wood blocks at a site with rapid initial decomposition, and higher amounts remaining for three species at three colder sites. ¹³C NMR spectra obtained with rapid cross-polarization (CP) mainly showed increasing similarity among the foliar litters, although wood showed little change in composition. Foliage generally showed loss of O- and di-O-alkyl C, mainly from carbohydrate, and increase in alkyl, aromatic, phenolic and carboxyl C. However, O-alkyl C loss was limited, especially for litters with slow initial decomposition, and many litters showed relatively small changes in intensity distribution. Quantitative ¹³C (“BD”) spectra showed similar trends, but even smaller changes in C composition, and 6-year CP difference spectra showed that C was lost across the whole range of structures. Changes in δ¹³C were small and variable, but could be correlated to some extent with loss of carbohydrates versus tannins. Lignin was not selectively preserved, and the increase of resistant structures derived from lignin, tannins, and cutin collectively accounts for increasing AUR. Compositional changes of NMR C fractions across sites with different temperatures were small and inconsistent, likely due to the influence of other site factors; however, changes in their contents did largely follow consistent trajectories with %C remaining.     

Insecticidal and Repellent Activities of the Essential Oil of Callistemon citrinus (Myrtaceae) Against Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

The essential oil of Callistemon citrinus (Curtis) leaves was extracted by hydro distillation and tested on female and male adults of Callosobruchus macullatus (F.) for insecticidal and repellent effects. GC-MS analysis was used to identify and quantify the volatile composition of the essential oil. Results showed that 1,8-cineole (34.2%) and α-pinene (29.0%) were the major components of the oil. Callistemon citrinus oil was found to be toxic to adult insects when applied by fumigation. Responses varied according to the gender of the insect and exposure time. LC₅₀ values were 12.88 and 84.4 μL.?L^?1 for males and females, respectively. An increase in exposure time from 3 to 24 h caused an increase in mortality from 50% to 100% in males and from 15.5% to 85.2% in females, at the highest concentration (500 μL.?L^?1). The essential oil also had a repellent effect against C. macullatus in a filter paper arena test. After 2 and 4 h, 86% and 94%, respectively, repellent effects were demonstrated at the highest concentration of 0.4 μL?.cm^?2. These observations suggest that C. citrinus essential oil may be usefully applied to control storage pests.     

四种类型栓皮栎栲胶含量

为了阐明不同类型(薄皮深裂、薄皮浅裂、厚皮深裂、厚皮浅裂)栓皮栎各器官栲胶含量,系统采集了秦岭南坡商州区4种类型的栓皮栎不同径级植株根、主干木材、叶片、枝条和树皮样品,用紫外光分光光度法(UV Spectrophotometry)测定了4种类型栓皮栎不同器官栲胶含量,分析了林木生长与栲胶含量的关系。结果表明,(1)4种类型栓皮栎的根、主干木材、叶片、枝条和树皮单宁含量平均值分别为2.64%—3.02%、1.74%—2.02%、6.63%—7.21%、4.69%—5.10%、8.87%—9.46%,叶片、枝条和树皮均可以作为栲胶生产主要原材料。(2)不同类型各器官单宁含量存在极显著差异(P0.01),4种类型的树皮、叶片、枝条单宁含量均表现为:厚皮深裂型厚皮浅裂型薄皮深裂型薄皮浅裂型;根部和主干木材在4种类型间单宁含量表现为:薄皮深裂型薄皮浅裂型厚皮深裂型厚皮浅裂型;厚皮深裂类型的栓皮栎是提取栲胶的最佳类型。(3)4种类型栓皮栎各器官单宁含量与胸径呈显著正相关,各器官单宁含量随着栓皮栎胸径增大而增多,并最终趋于稳定,在第Ⅳ径级(胸径20.1—25cm)时,栲胶含量均值最大。因此,栲胶生产可以高效利用第Ⅳ径级及其以上径级的栓皮栎,并对第Ⅳ径级以下栓皮栎进行定向培育和重点保护。     

Compositional and Thermal Evaluation of Lignocellulosic and Poultry Litter Chars via High and Low Temperature Pyrolysis

Inorganic elements in biomass feedstocks can influence thermochemical reactions as well as the resultant char’s elemental, compositional, and thermal characteristics. Chars were produced using slow pyrolysis under low (≤400°C) and high (≥500°C) temperature regimes from sugarcane bagasse, peanut hulls, pecan shell, pine chips, poultry litter, and switchgrass. The chars and raw feedstocks were characterized for their elemental, structural, and thermal properties to ascertain the implications of feedstock selection and pyrolysis temperatures on these properties. Char mass yields from the six feedstocks ranged between 28% and 78% by weight while carbon yields ranged between 44% and 89%. In both instances, lower yields were obtained with increasing pyrolysis temperature. Higher pyrolysis temperatures (≥500°C) resulted in more neutral to alkaline chars possessing greater ash contents and increased aromatic character with narrow O/C and H/C ratios. A significant exponential curve response (r ²?=?0.87, P?<?0.001) was revealed between char mass yields vs. pyrolysis temperature. All raw feedstocks and chars contained mixed amounts of macro-, micro-, and trace element concentrations. The higher heating values (HHV) tended to increase with heightened pyrolysis temperature with some chars producing >30 MJ kg^?1. The chars’ HHV values inversely correlated to their total ash and Cl content. Lignocelluloses chars had better thermal characteristics and lower ash quality concerns implying suitable service in thermal energy production. In contrast, poultry litter char had greater ash contents, medium HHV values, and contained corrosive inorganic elements, which rendered it problematic as a feedstock for thermal energy generation.     

Induction of conifer immune responses by phytopathogenic fungus metabolites

The elicitor activity of compounds extracted from the mycelia of six species of phytopathogenic fungi was assessed from the sizes of necrotic lesions on the external surface of the living trunk phloem of five coniferous species inhabiting Siberia: Siberian larch (Larix sibirica L.), Scotch pine (Pinus sylvestris L.), Siberian spruce (Picea obovata Ledeb.), Siberian fir (Abies sibirica L.), and cedar pine (Pinus sibirica (Rupr.) Mayr.). The compounds for inoculation were extracted from the mycelium of ascomycetes imperfect, and basidium fungi; the living mycelia of these fungi were also used. The fungal extract or mycelium was placed into the hollows 7 mm in diameter in the trunk bark. Infection triggered the formation of hypersensitivity necrotic lesions in the inner bark exceeding in size those appeared after control wounding of four tree species (larch, pine, spruce, and cedar); fir was an exclusion. In experiments with tree trunks and conifer calluses, a dependence of immune response parameters (the sizes of necrotic lesions and the content of lignin and bound proanthocyanidins) on the quantity of the fungal preparation was elucidated. The largest necrotic lesions appeared after injection of 500μg of the fungal preparation into the hollow in the trunk, and its higher quantities did not increase the indices measured. The size of the necrotic lesion on the trunk bark is supposed to be used as a promising index characterizing the level of tree immunity and tolerance under various ecological conditions.     

N2-Fixation and Seedling Growth Promotion of Lodgepole Pine by Endophytic Paenibacillus polymyxa

We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus polymyxa P2b-2R, a diazotrophic bacterium previously isolated from internal stem tissue of a naturally regenerating pine seedling to evaluate biological nitrogen fixation and seedling growth promotion by this microorganism. Seedlings generated from pine seed inoculated with strain P2b-2R were grown for up to 13 months in a N-limited soil mix containing 0.7 mM available N labeled as Ca(¹⁵NO₃)₂ to facilitate detection of N₂-fixation. Strain P2b-2R developed a persistent endophytic population comprising 10²–10⁶?cfu?g^?1 plant tissue inside pine roots, stems, and needles during the experiment. At the end of the growth period, P2b-2R had reduced seedling mortality by 14 % and ¹⁵N foliar N abundance 79 % and doubled foliar N concentration and seedling biomass compared to controls. Our results suggest that N₂-fixation by P. polymyxa enhanced growth of pine seedlings and support the hypothesis that plant-associated diazotrophs capable of endophytic colonization can satisfy a significant proportion of the N required by tree seedlings growing under N-limited conditions.