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.     

Structural changes of corn stover lignin during acid pretreatment

In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70?% at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by (31)P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180?°C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.     

Quantitative solid state NMR analysis of residues from acid hydrolysis of loblolly pine wood

The composition of solid residues from hydrolysis reactions of loblolly pine wood with dilute mineral acids is analyzed by ¹³C Cross Polarization Magic Angle Spinning (CP MAS) NMR spectroscopy. Using this method, the carbohydrate and lignin fractions are quantified in less than 3 h as compared to over a day using wet chemical methods. In addition to the quantitative information, ¹³C CP MAS NMR spectroscopy provides information on the formation of additional extractives and pseudo lignin from the carbohydrates. Being a non-destructive technique, NMR spectroscopy provides unambiguous evidence of the presence of side reactions and products, which is a clear advantage over the wet chemical analytical methods. Quantitative results from NMR spectroscopy and proximate analysis are compared for the residues from hydrolysis of loblolly pine wood under 13 different conditions; samples were treated either at 150 °C or 200 °C in the presence of various acids (HCl, H₂SO₄, H₃PO₄, HNO₃ and TFA) or water. The lignin content determined by both methods differed on averaged by 2.9 wt% resulting in a standard deviation of 3.5 wt%. It is shown that solid degradation products are formed from saccharide precursors under harsh reaction conditions. These degradation reactions limit the total possible yield of monosaccharides from any subsequent reaction.     

Phenol and phenolics from lignocellulosic biomass by catalytic microwave pyrolysis

Catalytic microwave pyrolysis of biomass using activated carbon was investigated to determine the effects of pyrolytic conditions on the yields of phenol and phenolics. The high concentrations of phenol (38.9%) and phenolics (66.9%) were obtained at the temperature of 589 K, catalyst-to-biomass ratio of 3:1 and retention time of 8 min. The increase of phenol and its derivatives compared to pyrolysis without catalysts has a close relationship with the decomposition of lignin under the performance of activated carbon. The concentration of esters was also increased using activated carbon as a catalyst. The high content of phenols obtained in this study can be used either directly as fuel after upgrading or as feedstock of bio-based phenols for chemical industry.     

Characterization of carbons derived from cellulose and lignin and their oxidative behavior

In this study the oxidative behavior of carbons derived from cellulose and lignin were compared using thermogravimetric analysis (TGA). Specific surface area and chemical composition of the two types of carbon were analyzed using nitrogen adsorption at 77 K and infrared spectroscopy respectively. The results demonstrate that cellulose carbon has a higher reaction order and lower activation energy than lignin carbon under identical experimental conditions when they were prepared at temperatures lower than 500 °C. However, such differences were considerably reduced for the carbon samples prepared at temperatures greater than 700 °C. It was verified that lignin carbon is more stable than cellulose carbon due to its higher content of aromatic structures when they are prepared at lower temperature. The specific surface area and porosity have a more limited contribution to the differential oxidative behaviors of the two types of carbon. This research has significance related to the formation of carbon nanotubes from plant materials during low temperature carbonization.     

Microbial pretreatment of cotton stalks by submerged cultivation of Phanerochaete chrysosporium

This study used the fungus, Phanerochaete chrysosporium, to pretreat cotton stalks with two methods, shallow stationary and agitated cultivation, at three supplemental salt concentrations. Pretreatment efficiencies were compared by evaluating lignin degradation, solid recovery and carbohydrate availability over a 14-day period. Shallow stationary cultivation with no salts gave 20.7% lignin degradation along with 76.3% solid recovery and 29.0% carbohydrate availability. The highest lignin degradation of 33.9% at a corresponding solid recovery and carbohydrate availability of 67.8% and 18.4%, respectively, was obtained through agitated cultivation with Modified NREL salts. Cultivation beyond 10 days did not significantly increase lignin degradation during 14 days of pretreatment. Manganese addition during shallow stationary and agitated cultivation resulted in higher solid recoveries of over 80% but lower lignin degradation. Although agitated cultivation resulted in better delignification, results indicate that pretreatment under submerged shallow stationary conditions provides a better balance between lignin degradation and carbohydrate availability.     

Woody biomass: Niche position as a source of sustainable renewable chemicals and energy and kinetics of hot-water extraction/hydrolysis

The conversion of biomass to chemicals and energy is imperative to sustaining our way of life as known to us today. Fossil chemical and energy sources are traditionally regarded as wastes from a distant past. Petroleum, natural gas, and coal are not being regenerated in a sustainable manner. However, biomass sources such as algae, grasses, bushes and forests are continuously being replenished. Woody biomass represents the most abundant and available biomass source. Woody biomass is a reliably sustainable source of chemicals and energy that could be replenished at a rate consistent with our needs. The biorefinery is a concept describing the collection of processes used to convert biomass to chemicals and energy. Woody biomass presents more challenges than cereal grains for conversion to platform chemicals due to its stereochemical structures. Woody biomass can be thought of as comprised of at least four components: extractives, hemicellulose, lignin and cellulose. Each of these four components has a different degree of resistance to chemical, thermal and biological degradation. The biorefinery concept proposed at ESF (State University of New York — College of Environmental Science and Forestry) aims at incremental sequential deconstruction, fractionation/conversion of woody biomass to achieve efficient separation of major components. The emphasis of this work is on the kinetics of hot-water extraction, filling the gap in the fundamental understanding, linking engineering developments, and completing the first step in the biorefinery processes. This first step removes extractives and hemicellulose fractions from woody biomass. While extractives and hemicellulose are largely removed in the extraction liquor, cellulose and lignin largely remain in the residual woody structure. Xylo-oligomers and acetic acid in the extract are the major components having the greatest potential value for development. Extraction/hydrolysis involves at least 16 general reactions that could be divided into four categories: adsorption of proton onto woody biomass, hydrolysis reactions on the woody biomass surface, dissolution of soluble substances into the extraction liquor, and hydrolysis and dehydration decomposition in the extraction liquor. The extraction/hydrolysis rates are significantly simplified when the reactivity of all the intermonomer bonds are regarded as identical within each macromolecule, and the overall reactivity are identical for all the extractable macromolecules on the surface. A pseudo-first order extraction rate expression has been derived based on concentrations in monomer units. The reaction rate constant is however lower at the beginning of the extraction than that towards the end of the extraction. Furthermore, the H-factor and/or severity factor can be applied to lump the effects of temperature and residence time on the extraction process, at least for short times. This provides a means to control and optimize the performance of the extraction process effectively.     

Kinetic study of recycled newspaper liquefaction in polyol solvent

The kinetics of liquefaction of recycled newspaper was investigated. Liquefaction was carried out in the presence of polyhydric alcohols and ethylene carbonate under acidic conditions. The liquefaction yield was found to be a function of the solvent type, sulfuric acid concentration, temperature, and the ratio of solvent to newspaper. The total liquefaction rate and degradation rates of cellulose, hemicelluloses, and lignin, which are the major components of newspaper, were measured. From the results, it was found that the total liquefaction rate did not follow first order reaction kinetics, because of the difference in the degradation rate of each component and the condensation of lignin. The degradation rates of lignin and hemicellulose were approximately 13 and 7 times greater than that of cellulose, respectively.     

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.     

木质素降解菌BYL-7的筛选及降解条件优化

【背景】微生物降解木质素因其具有降解效率高和环保等特点而备受关注。【目的】筛选高效木质素降解真菌,并对其降解条件进行优化。【方法】通过愈创木酚-马铃薯葡萄糖琼脂(potato dextrose agar,PDA)和苯胺蓝平板法筛选高效木质素降解菌株,利用单因素筛选及响应面试验对培养条件进行优化。【结果】筛选到一株高效木质素降解菌BYL-7,经形态和多序列分析初步确定为Trametes versicolor。单因素试验证明初始pH、温度和接种量为降解木质素显著影响因子,响应面试验确定降解木质素最优条件为：初始pH 6.7,温度25 °C,接种量8%。在此条件下,碱性木质素降解率为36.5%,比未优化前提高54.0%;水稻秸秆木质素、半纤维素和纤维素降解率分别为32.8%、21.5%、13.2%,其中木质素降解率比未优化前提高36.1%;漆酶活性在第6天达到峰值120.0 U/L,比未优化前提高25.0%;木质素过氧化物酶活性在第6天达到峰值1 343.8 U/L,比未优化前提高36.0%;锰过氧化物酶活性在第5天达到峰值463.8 U/L,比未优化前提高31.7%。【结论】研究结果为木质素的降解提供了良好的菌种资源,同时也为后续木质素的研究积累了相关数据。     

Improved pretreatment of lignocellulosic biomass using enzymatically-generated peracetic acid

Release of sugars from lignocellulosic biomass is inefficient because lignin, an aromatic polymer, blocks access of enzymes to the sugar polymers. Pretreatments remove lignin and disrupt its structure, thereby enhancing sugar release. In previous work, enzymatically generated peracetic acid was used to pretreat aspen wood. This pretreatment removed 45% of the lignin and the subsequent saccharification released 97% of the sugars remaining after pretreatment. In this paper, the amount of enzyme needed is reduced tenfold using first, an improved enzyme variant that makes twice as much peracetic acid and second, a two-phase reaction to generate the peracetic acid, which allows enzyme reuse. In addition, the eight pretreatment cycles are reduced to only one by increasing the volume of peracetic acid solution and increasing the temperature to 60 °C and the reaction time to 6 h. For the pretreatment step, the weight ratio of peracetic acid to wood determines the amount of lignin removed.     

The influence of lignin content and temperature on the biodegradation of lignocellulose in composting conditions

The aim of this research was to study the influence of lignin content and composting temperature on the biodegradation of lignin-containing pulp and paper products in a controlled composting test (European standard prEN 14046). Lignin reduced the biodegradation of the samples, and there was a linear correlation between the lignin content and the biodegradation of pulp and paper products at 58°C. The influence of incubation temperature (35, 50 and 58°C) on biodegradation was studied using bleached kraft paper containing 0.2 wt% lignin and mechanical pulp (stone-ground wood) containing 24–27 wt% lignin. Mechanical pulp biodegraded better at lower temperatures, while kraft paper biodegraded well at all three temperatures. Microbial activity was evaluated by measuring CO₂ evolution and the change in ATP content, and fungal biomass by measuring the ergosterol content during the composting experiments. Kraft paper strongly increased microbial activity during the controlled composting test, but the activity returned to the background level at the end of the composting test. The proportion of sample carbon converted to microbial biomass carbon was considerably higher at lower incubation temperatures. Changes in microbial community structure during biodegradation of mechanical pulp and kraft paper at 50°C were studied by the PCR-based technique denaturing gradient gel electrophoresis. Changes in the microbial community were observed during the intensive degradation phase of kraft paper. Electronic Publication     

Lignocellulosic biomass fractionation: I - solvent extraction in a novel reactor

Capium (elephant grass) chips were treated at 180°C for 1 to 3 h, solid concentrations 3 to 14%, with 50% aquous ethanol in a recently-developed reactor. About 70% delignification and 90% hemicellulose removal in solid form, and 95% glucose yield in 16h by enzymatic hydrolysis, were obtained under optimum reaction conditions. The effectiveness of lignocellulosic biomass fractionation is due largely to the reactor design which facilitates efficient separation of components and overcomes lignin condensation problems.     

Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw

Wheat and rye straws were pretreated with ozone to increase the enzymatic hydrolysis extent of potentially fermentable sugars. Through a 2(5-1) factorial design, this work studies the influence of five operating parameters (moisture content, particle size, ozone concentration, type of biomass and air/ozone flow rate) on ozonization pretreatment of straw in a fixed bed reactor under room conditions. The acid insoluble lignin content of the biomass was reduced in all experiments involving hemicellulose degradation. Near negligible losses of cellulose were observed. Enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16% in non-ozonated wheat and rye straw respectively. Moisture content and type of biomass showed the most significant effects on ozonolysis. Additionally, ozonolysis experiments in basic medium with sodium hydroxide evidenced a reduction in solubilization and/or degradation of lignin and reliable cellulose and hemicellulose degradation.     

The Role of Submarine Hydrothermal Systems in the Synthesis of Amino Acids

There is little consensus regarding the plausibility of organic synthesis in submarine hydrothermal systems (SHSs) and its possible relevance to the origin of life. The primary reason for the persistence of this debate is that most experimental high temperature and high-pressure organic synthesis studies have neglected important geochemical constraints with respect to source material composition. We report here the results of experiments exploring the potential for amino acid synthesis at high temperature from synthetic seawater solutions of varying composition. The synthesis of amino acids was examined as a function of temperature, heating time, starting material composition and concentration. Using very favorable reactant conditions (high concentrations of reactive, reduced species), small amounts of a limited set of amino acids are generated at moderate temperature conditions (∼125–175°C) over short heating times of a few days, but even these products are significantly decomposed after exposure times of approximately 1 week. The high concentration dependence observed for these synthetic reactions are demonstrated by the fact that a 10-fold drop in concentration results in orders of magnitude lower yields of amino acids. There may be other synthetic mechanisms not studied herein that merit investigation, but the results are likely to be similar. We conclude that although amino acids can be generated from simple likely environmentally available precursors under SHS conditions, the equilibrium at high temperatures characteristic of SHSs favors net amino acid degradation rather than synthesis, and that synthesis at lower temperatures may be more favorable.     

Effects of spray-drying and storage on astaxanthin content of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> biomass

The main objective of this study was to evaluate the stability of astaxanthin after drying and storage at different conditions during a 9-week period. Recovery of astaxanthin was evaluated by extracting pigments from the dried powders and analysing extracts by HPLC. The powders obtained were stored under different conditions of temperature and oxygen level and the effects on the degradation of astaxanthin were examined. Under the experimental conditions conducted in this study, the drying temperature that yielded the highest content of astaxanthin was 220°C, as the inlet, and 120°C, as the outlet temperature of the drying chamber. The best results were obtained for biomass dried at 180/110°C and stored at −21°C under nitrogen, with astaxanthin degradation lower than 10% after 9 weeks of storage. A reasonable preservation of astaxanthin can be achieved by conditions 180/80°C, −21°C nitrogen, 180/110°C, 21°C nitrogen, and 220/80°C, 21°C vacuum: the ratio of astaxanthin degradation is equal or inferior to 40%. In order to prevent astaxanthin degradation of Haematococcus pluvialis biomass, it is recommended the storage of the spray dried carotenized cells (180/110oC) under nitrogen and −21°C.     

Direct biosynthesis of adipic acid from lignin-derived aromatics using engineered Pseudomonas putida KT2440

Lignin is one largely untapped natural resource that can be exploited as a raw material for the bioproduction of value-added chemicals. Meanwhile, the current petroleum-based process for the production of adipic acid faces sustainability challenges. Here we report the successful engineering of Pseudomonas putida KT2440 strain for the direct biosynthesis of adipic acid from lignin-derived aromatics. The devised bio-adipic acid route features an artificial biosynthetic pathway that is connected to the endogenous aromatics degradation pathway of the host at the branching point, 3-ketoadipoyl-CoA, by taking advantage of the unique carbon skeleton of this key intermediate. Studies of the metabolism of 3-ketoadipoyl-CoA led to the discovery of crosstalk between two aromatics degradation pathways in KT2440. This knowledge facilitated the formulation and implementation of metabolic engineering strategies to optimize the carbon flux into the biosynthesis of adipic acid. By optimizing pathway expression and cultivation conditions, an engineered strain AA-1 produced adipic acid at 0.76 g/L and 18.4% molar yield under shake-flask conditions and 2.5 g/L and 17.4% molar yield under fermenter-controlled conditions from common aromatics that can be derived from lignin. This represents the first example of the direct adipic acid production from model compounds of lignin depolymerization.     

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment.     

Thermal degradation mechanisms of wood under inert and oxidative environments using DAEM methods

The pyrolytic behavior of wood is investigated under inert and oxidative conditions. The TGA experiment is given a temperature variation from 323 to 1173 K by setting the heating rate between 5 and 40 K/min. The results of DTG curves show that the hemicellulose shoulder peak for birch is more visible under inert atmosphere due to the higher content of reactive xylan-based hemicellulose (mannan-based for pine). When oxygen presents, thermal reactivity of biomass (especially the cellulose) is greatly enhanced due to the acceleration of mass loss in the first stage, and complex reactions occur simultaneously in the second stage when char and lignin oxidize. A new kinetic model is employed for biomass pyrolysis, namely the distributed activation energy model (DAEM). Under inert atmosphere, the distributed activation energy for the two species is found to be increased from 180 to 220 kJ/mol at the solid conversion of 10-85% with the high correlation coefficient. Under oxidative atmosphere, the distributed activation energy is about 175-235 kJ/mol at the solid conversion of 10-65% and 300-770 kJ/mol at the solid conversion of 70-95% with the low correlation coefficient (below 0.90). Comparatively, the activation energy obtained from established global kinetic model is correspondingly lower than that from DAEM under both inert and oxidative environments, giving relatively higher correlation coefficient (more than 0.96). The results imply that the DAEM is not suitable for oxidative pyrolysis of biomass (especially for the second mass loss stage in air), but it could represent the intrinsic mechanism of thermal decomposition of wood under nitrogen better than global kinetic model when it is applicable.