Cyanogenic Residues: Environmental Impacts,Complexation with Humic Substances,and Possible Application as Biofertilizer

Treated and untreated rice straw extensively exists in the soil. In order to elucidate its possible effect on the fate of organic pollutants, sorption of pyrene by rice straw and its main constituents (lignin, cellulose, and hemi-cellulose) were studied, as single solute and in the presence of other co-existing organic pollutants, phenanthrene (Phen), benzo[a]pyrene (BaP), phenol, and pentachlorophenol (PCP). Pyrene showed the greatest sorption on lignin with greater aromaticity and smaller polarity, and the sorption coefficient was almost two orders of magnitude greater than those on cellulose and hemi-cellulose. Bi-solute sorption results showed that Phen, BaP and PCP exhibited apparent competitive sorption with pyrene on the four sorbents; while the existence of phenol promoted the sorption of pyrene on rice straw and lignin but inhibited the sorption on cellulose and hemi-cellulose. For the two polycyclic aromatic hydrocarbon (PAH) co-solutes and PCP, hydrophobicity and molecular size played important roles in competition, suggesting the direct competition for hydrophobic sorption sites and pore blockage mechanisms. In contrast, the polar co-solute, phenol showed different effects on pyrene sorption onto the four sorbents, suggesting that multiple interactions between polar organic compounds and sorbents are involved in the sorption.     

Effect of different wood pretreatments on the sorption-desorption of linuron and metalaxyl by woods

The sorption-desorption of two different pesticides, linuron and metalaxyl, by woods was studied. Sorbent/solution ratio and sorption kinetics were also determined. Untreated wood and water, NaOH, HCl, and octadecyltrimethylammonium bromide (ODTMA) treated pine (softwood) and oak (hardwood) were used as sorbents. Linuron and metalaxyl were sorbed by untreated woods up to 80 and 40%, respectively, in a short time when the sorbent/solution ratio of 1:10 was used. Sorption of pesticides was significantly higher by pine, having higher lignin content, than by oak. Freundlich sorption constants (K(f)) were 96.2 and 74.4 (linuron) and 8.28 and 4.95 (metalaxyl) for untreated pine and oak woods and increased 1.04-2.35-fold (linuron) and 1.33-2.17-fold (metalaxyl) when woods were treated. The sorption was higher by HCl- and ODTMA-treated woods. Additionally, Freundlich desorption constants also indicated greater sorption irreversibility of both pesticides for treated woods than for untreated woods. The results revealed wood residues as a promising, low-cost, and environmentally friendly material to immobilize pesticides in soils, preventing water contamination. Wood treatments aimed at removing soluble wood extracts or at modifying wood chemical structure could increase their sorption capacity.     

Sorption behavior of volatile phenols at the oak wood/wine interface in a model system

The sorption in a model system of aroma compounds of enological interest (mixture of the eight derivatives from guaiacol, 4-ethylphenol, and whiskylactone) onto wood was investigated to assess the influence of wood on the concentration of these volatiles during the aging of wine. To evaluate the influence of the solubility of aroma compounds in sorption phenomena, this parameter was determined for each volatile compound in model wine at 10 and 25 degrees C. The solubility is significantly higher in the model wine than in water and remains constant in the range of temperatures studied, except for guaiacol and vanillin. Kinetic and equilibrium sorptions were investigated. Sorption kinetics showed that the sorption equilibrium for all aroma compounds was reached after 6-7 days. From the study of the individual sorption isotherms, two distinct kinds of sorption behavior were observed depending on the presence or not of an ethylenic para substituent conjugated to the phenyl ring. K(ww) partition coefficients between the wood and the model wine were determined, which exhibited an unusual positive variation with temperature.     

城市污泥与调理剂混合堆肥过程中有机质组分的变化

【目的】研究城市污泥堆肥过程中各项有机质组分及碳、 氮在堆肥过程中的形成与转化,以期改善堆肥的生物有效性,促进其土地利用。【方法】在工厂规模化下,以城市污泥、 蘑菇渣锯末以及返混料按照6∶3∶1的质量比混合形成堆肥物料,辅以强制通风措施和翻抛,进行为期18 d的高温堆肥试验。堆肥期间定期采样,测定指标包括温度、 C/N值、 pH、 含水率、 有机质降解率、 水溶性组分、 半纤维素、 纤维素和木质素,研究堆肥期间不同阶段堆肥物料中有机质组分的动态变化。【结果】堆体温度随着发酵时间的延长呈现先升高后降低的趋势,最高温度达到71.3℃; 含水率由60.7%降低到51.4%,pH呈现先升高后降低的趋势,总体处于6.0~7.5之间; 总有机碳含量持续下降,氮素含量表现为高温期持续下降随后呈上升的趋势; 初始阶段,堆肥物料中四种成分含量分布为: 水溶性组分纤维素半纤维素木质素,至堆肥结束变化为: 纤维素水溶性组分木质素半纤维素,经过堆肥之后水溶性组分及半纤维素含量分别由39.5%和20.1%下降为27.9%和14.4%,纤维素含量由初始的21.8%上升至29.5%,木质素含量相对稳定不变。物料经过堆肥化处理后达到腐熟标准,水溶性组分和半纤维素含量分别降低了38.6%和38.8%,纤维素和木质素含量在高温期分别降解了11.7%和18.5%; 物料总量降低了9.8%。水溶性组分的主要降解阶段为高温期,期间降解部分占总降解量的65.5%; 半纤维素的主要降解阶段为稳定期,稳定期降解部分占总降解量的69.1%,且有继续降解的趋势; 纤维素和木质素仅在高温期有少量降解; 氮素则表现为高温期铵态氮的损失及稳定期硝态氮的积累。【结论】堆肥化处理在实现污泥减量化基础上,污泥中有机质得到了稳定化,有利于城市污泥的土地利用。     

Effects of increased nitrogen supply on the lignification of poplar wood

The short-term influence of adequate and high nitrogen fertilization on poplar lignification was investigated. The high nitrogen supply decreased lignin staining in the newly formed secondary xylem, indicating that lignin deposition was affected. Acetyl bromide determinations gave a 9-10% decrease in lignin content; however, Klason lignin content was unchanged. Thioacidolysis showed that elevated N supply affected lignin structure such that there was a reduced frequency of lignin units involved in beta-O-4 bonds, a reduced syringyl/guaiacyl ratio, an increased frequency of p-hydroxyphenyl lignin units, more guaiacyl units with free phenolic groups, and more p-hydroxybenzoic acid ester-linked to poplar lignins. These features suggest that lignins from poplars grown under high N bear structural similarities to lignins formed during early stages of wood development. The findings also indicate that a gravitational stimulus inducing the formation of tension wood and high N availability lead to similar and additive effects on lignin content and structure.     

Impact of plant cell wall network on biodegradation in soil: Role of lignin composition and phenolic acids in roots from 16 maize genotypes

Residue quality is a key factor governing biodegradation and the fate of C in soil. Most investigations of relationships existing between crop residue quality and soil decomposition have been based on determining the relative proportions of soluble, cellulose, hemicellulose and lignin components. However, cell wall cohesion is increased by tight interconnections between polysaccharides and lignin that involve cross-linking agents (phenolic acids). The aim of this study was to determine the role of lignin composition and phenolic acids on short- to medium-term decomposition of maize roots in soil. Sixteen maize genotypes, presenting a range of chemical characteristics related to root lignin and phenolic acids, were used. The main components were characterized by Van Soest (VS) extraction and cell wall acid hydrolysis, and the non-condensed Syringyl and Guaicyl lignin monomers, esterified phenolic acids and etherified phenolic acids were determined. Maize roots were then incubated in soil under controlled conditions (15 °C, −80 kPa moisture) for 796 days. Results showed that VS extraction over-estimated the structural hemicellulose content and that VS lignin was more recalcitrant than Klason lignin. The tremendous effect of cell wall chemical characteristics was shown by marked variations (almost two-fold differences in C mineralization), between the 16 maize roots. Decomposition was controlled by soluble residue components in the short term whereas lignin and the interconnections between cell wall polymers were important in the long-term. Notably the cell wall domain rich in non-condensed lignin and esterified phenolic acids was prone to decomposition whereas the presence of etherified ferulic acids seemed to hamper cell wall decomposition.     

木质素对灵芝菌丝体生长的影响

为了解添加外源木质素对灵芝菌丝体生长的影响,该研究通过在灵芝培养基中添加纤维素、半纤维素和木质素,考察了外源添加木质纤维素主要组分对灵芝菌体生长的影响,发现木质素添加明显促进灵芝菌丝体生长;进一步考察添加不同浓度木质素灵芝菌丝体生长情况以及灵芝产多糖、三萜变化规律。结果表明少量添加木质素对灵芝菌丝体生长有明显促进作用,木质素加量为1.0%时,灵芝菌丝体生长情况最好,生物量比对照提高了56%,同时灵芝胞外多糖、胞内多糖及灵芝胞外三萜、胞内三萜的合成均有明显提高,其中胞内多糖提高43%;4种灵芝菌株发酵产多糖、三萜结果对比分析表明1.0%木质素添加均能提高不同菌株发酵多糖、三萜含量,木质素作为营养基质在不同灵芝菌株培养过程中添加均能促进菌丝体活性物质合成。研究结果可为灵芝菌生长代谢研究及人工栽培提供科学依据和参考。     

Toward a better understanding of the lignin isolation process from wood

The recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) coupled with the novel combination of derivatization followed by reductive cleavage (DFRC) and quantitative (31)P NMR spectroscopy were used to better understand the lignin isolation process from wood. The EMAL protocol is shown to offer access at lignin samples that are more representative of the overall lignin present in milled wood. The combination of DFRC/(31)P NMR provided a detailed picture on the effects of the isolation conditions on the lignin structure. More specifically, we have used vibratory and ball milling as the two methods of wood pulverization and have compared their effects on the lignin structures and molecular weights. Vibratory-milling conditions cause substantial lignin depolymerization. Lignin depolymerization occurs via the cleavage of uncondensed beta-aryl ether linkages, while condensed beta-aryl ethers and dibenzodioxocins were found to be resistant to such mechanical action. Condensation and side chain oxidations were induced mechanochemically under vibratory-milling conditions as evidenced by the increased amounts of condensed phenolic hydroxyl and carboxylic acid groups. Alternatively, the mild mechanical treatment offered by ball milling was found not to affect the isolated lignin macromolecular structure. However, the overall lignin yields were found to be compromised when the mechanical action was less intense, necessitating longer milling times under ball-milling conditions. As compared to other lignin preparations isolated from the same batch of milled wood, the yield of EMAL was about four times greater than the corresponding milled wood lignin (MWL) and about two times greater as compared to cellulolytic enzyme lignin (CEL). Molecular weight distribution analyses also pointed out that the EMAL protocol allows the isolation of lignin fractions that are not accessed by any other lignin isolation procedures.     

Comparative evaluation of three lignin isolation protocols for various wood species

Milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and enzymatic mild acidolysis lignin (EMAL) were isolated from different wood species and characterized by various techniques. The EMAL protocol offered gravimetric lignin yields 2-5 times greater than those of the corresponding MWL and CEL. The purities of the EMALs were 3.75-10.6% higher than those of their corresponding CELs, depending upon the wood species from which they were isolated. Molecular weight analyses showed that the EMAL protocol isolates lignin fractions that are not accessed by the other procedures evaluated, while 31P NMR spectroscopy revealed that MWL is more condensed and bears more phenolic hydroxyl groups than EMAL and CEL. The yields and purities of EMAL, MWL, and CEL from hardwood were greater than those obtained for the examined softwoods. Structural details obtained by DFRC (derivatization followed by reductive cleavage)/31P NMR revealed different contents of condensed and uncondensed beta-O-aryl ether structures, dibenzodioxocins, and condensed and uncondensed phenolic hydroxyl and carboxylic acid groups within lignins isolated from different wood species.     

根系主要成分含量对根系固土效能的影响

选取中国北方两种常见树种油松(Pinus tabulaeformis)和元宝枫(Acer truncatum),对这两种树0.2~6mm直径的根系直径、根系抗拉强度、整根的根土复合体强度、根系3种主要成分含量(纤维素含量、半纤维素含量与木质素含量)进行测定。结果表明,随着根系直径的增加,植物根系半纤维素含量增高,纤维素含量与木质素含量降低,从而植物的抗拉强度降低。植物根系抗拉强度与纤维素含量、木质素含量成正相关关系,与半纤维素含量成负相关关系。因此,在排除根系结构影响的前提下,植物根系纤维素含量与木质素含量是导致不同植物种固土效果差异的直接原因。     

NaOH预处理提高甘蔗叶产甲烷性能及其机理分析

为提高甘蔗叶厌氧消化的产甲烷性能,采用NaOH对粉碎后的甘蔗叶进行了预处理,得到了不同NaOH浓度、不同预处理时间条件下甘蔗叶厌氧消化的甲烷产率,并研究比较了预处理前后甘蔗叶微观物理形态、化学分子结构和化学组分的变化。结果表明:与未预处理甘蔗叶相比,NaOH预处理甘蔗叶的累计产甲烷量提高了22.02%～89.94%,厌氧消化时间T80缩短了2～4 d,其中6%NaOH-5d预处理甘蔗叶的产甲烷性能最好;NaOH破坏了甘蔗叶表面蜡质层和细胞壁结构,促进了甘蔗叶表面二氧化硅、木质素等分解,打破了对纤维素的束缚;预处理后甘蔗叶的木质纤维素结构发生明显变化,其中木质素的羟基、甲氧基和羰基等部分官能团发生不同程度断裂,紧致的大分子结构发生分解,纤维素的结晶度降低,部分氢键遭到破坏,半纤维素发生了分子间和分子内的降解;预处理甘蔗叶的木质纤维素含量均有不同程度的降低,可被微生物分解利用的有机物质增多,其中6%NaOH-5d预处理甘蔗叶厌氧消化的木质素、纤维素、半纤维素降解率分别提高了9.27%、25.14%和21.52%。因此NaOH预处理是一种提高甘蔗叶厌氧消化产甲烷性能的有效方法。     

Studies on the effect of ball milling on lignin structure using a modified DFRC method.

The structures of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and residual lignin (REL) from a loblolly pine were analyzed using a modified derivatization followed by reductive cleavage (DFRC) method developed to allow the quantitative determination of three different structural monomeric products originating in lignin: phenolic beta-O-4, alpha-O-4, and etherified beta-O-4 structures. Results show that MWL and CEL are structurally identical, with an increased phenolic beta-O-4 content compared to that of the original Wiley milled wood. These results indicate that the portion of lignin linked to carbohydrates and that not linked to carbohydrates are structurally the same. Modified DFRC analysis of the effect of ball milling on the structure of lignin in wood, MWL, CEL, and REL indicate that vibratory ball milling does not change the lignin structure provided certain precautions are taken. Specifically, dry vibratory ball milling under a nitrogen atmosphere causes substantial structural changes including condensation, whereas vibratory ball milling in toluene had little effect on the lignin structure. This indicates that the structural differences observed in MWL and CEL arise because of the extraction procedure, which preferentially extracts phenolic lignin structures. MWL and CEL are representative of the total lignin in wood; however, due primarily to the solvent extraction process, higher phenolic hydroxyl contents are observed. Nitrobenzene oxidation showed structural results similar to those from the modified DFRC method.     

Dissolution of wood in ionic liquids

The present paper demonstrates that both hardwoods and softwoods are readily soluble in various imidazolium-based ionic liquids (ILs) under gentle conditions. More specifically, a variety of ionic liquids can only partially dissolve wood chips, whereas ionic liquids such as 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride have good solvating power for Norway spruce sawdust and Norway spruce and Southern pine thermomechanical pulp (TMP) fibers. Despite the fact that the obtained solutions were not fully clear, these ionic liquids provided solutions which permitted the complete acetylation of the wood. Alternatively, transparent amber solutions of wood could be obtained when the dissolution of the same lignocellulosic samples was attempted in 1-benzyl-3-methylimidazolium chloride. This realization was based on a designed augmented interaction of the aromatic character of the cation of the ionic liquid with the lignin in the wood. After dissolution, wood can be regenerated as an amorphous mixture of its original components. The cellulose of the regenerated wood can be efficiently digested to glucose by a cellulase enzymatic hydrolysis treatment. Furthermore, completely acetylated wood was found to be readily soluble in chloroform, allowing, for the first time, detailed proton nuclear magnetic resonance (NMR) spectra and NMR diffusion measurements to be made. It was thus demonstrated that the dissolution of wood in ionic liquids now offers a variety of new possibilities for its structural and macromolecular characterization, without the prior isolation of its individual components. Furthermore, considering the relatively wide solubility and compatibility of ionic liquids with many organic or inorganic functional chemicals or polymers, it is envisaged that this research could create a variety of new strategies for converting abundant woody biomass to valuable biofuels, chemicals, and novel functional composite biomaterials.     

Estimation of wood density and chemical composition by means of diffuse reflectance mid-infrared Fourier transform (DRIFT-MIR) spectroscopy

Sitka spruce (Picea sitchensis) samples (491) from 50 different clones as well as 24 different tropical hardwoods and 20 Scots pine (Pinus sylvestris) samples were used to construct diffuse reflectance mid-infrared Fourier transform (DRIFT-MIR) based partial least squares (PLS) calibrations on lignin, cellulose, and wood resin contents and densities. Calibrations for density, lignin, and cellulose were established for all wood species combined into one data set as well as for the separate Sitka spruce data set. Relationships between wood resin and MIR data were constructed for the Sitka spruce data set as well as the combined Scots pine and Sitka spruce data sets. Calibrations containing only five wavenumbers instead of spectral ranges 4000-2800 and 1800-700 cm(-1) were also established. In addition, chemical factors contributing to wood density were studied. Chemical composition and density assessed from DRIFT-MIR calibrations had R2 and Q2 values in the ranges of 0.6-0.9 and 0.6-0.8, respectively. The PLS models gave residual mean squares error of prediction (RMSEP) values of 1.6-1.9, 2.8-3.7, and 0.4 for lignin, cellulose, and wood resin contents, respectively. Density test sets had RMSEP values ranging from 50 to 56. Reduced amount of wavenumbers can be utilized to predict the chemical composition and density of a wood, which should allow measurements of these properties using a hand-held device. MIR spectral data indicated that low-density samples had somewhat higher lignin contents than high-density samples. Correspondingly, high-density samples contained slightly more polysaccharides than low-density samples. This observation was consistent with the wet chemical data.     

杉木应压木形成中的显微特征及主要代谢成分变化

为了解析主要代谢成分与杉木(Cunninghamia lanceolata)应压木形成的关系,以3年生直立杉木苗为研究对象,与垂直方向倾斜45°放置30、60和90 d后观察其生长性状并测定木质纤维素含量,使用气相色谱-质谱联用仪(GC-MS)对木质部代谢成分进行鉴定和解析。解剖发现应压木木质部管胞发生变形;化学成分测定发现总木质素和半纤维素含量增加,纤维素含量降低;GC-MS检测出应压木、对应木和直立木中共有成分20种,其中有机酸7种、单糖3种、二糖2种、醇类3种、氨基酸2种、酚类和糖苷类各1种;主成分分析结合显著性检验发现,3个时期应压木和直立木中有11种共有差异代谢物,其中乙醇酸、甘氨酸、核糖酸和棕榈酸在应压木中的含量显著低于对应木和直立木,苹果酸、松醇、肌醇、半乳糖和蔗糖在应压木中的含量显著高于对应木和直立木。本试验结果从代谢角度解释了杉木应压木形成中的生物学变化,可为揭示木材形成的分子机制提供新途径,并为高品质木材的定向培育和木材功能性改良提供理论依据。     

Estimation and decomposition pattern of the lignin component in forest humus layers

Lignin is one of the most abundant polymeric organic constituents of forest litter. Due to its molecular structure and heterogeneity the isolation of lignin in an unchanged form and its exact determination in forest humus have not proved possible. The oxidative degradation with CuO provides a specific method for the characterization of intact lignin structures in forest humus layers. The sum of phenolic CuO oxidation products gives an overall pattern of lignin decomposition. The degree of alteration of the remnant lignin is described by the acid-to-aldehyde ratio (Ac:Al) of syringyl and vanillyl units for angiosperm and gymnosperm lignin and the ratio of syringyl-to-vanillyl units (S:V) for angiosperm lignin.The lignin component of three different forest humus layers investigated was partly decomposed. The residual lignin fraction had undergone extensive modification during microbial decomposition. The chemical changes occurring in the lignin molecule during decomposition in forest humus layers suggest similar mechanisms of lignin degradation (“white-rot”) are shared by wood and forest litter.     

Sodium amalgam reduction of humic acid—II. application of the method

The nature and distribution of the phenolic compounds found in humic acid on sodium amalgam reduction resemble those reported to be found in microbial cultures grown on lignifiedplanttissue.lt has also been shown that the more humified and decayed fractions of soil organic matter yield humic acids that bear less resemblance to lignin than do the humic acids from less decayed plant remains. The aromatic portion of humic acid would seem to consist of biologically modified and transformed lignin, together with phenolic units synthesized by soil microbes. Sodium amalgam reduction can be employed to estimate the degree of transformation, but cannot, however, be used to “finger print” humic acids.     

Aroma compound sorption by oak wood in a model wine

Oak wood used for wine barrels was immersed into a model wine containing eight aroma compounds (e.g., aromatic and terpene alcohols, ethyl esters, and aldehyde), for which activity coefficients in water and model wine were determined using the mutual solubility measurement. A mass balance of these volatiles considering their reactivity in model wine was established. For most of the studied aroma compounds, and mainly for linalool and ethyl octanoate, a sorption behavior into wood was reported for the first time. This phenomenon was selective and could not be related to the solubilities in model wine and hydrophobicities of the studied aroma compounds, suggesting that acid-base and polar characteristics of wood were more involved in this sorption mechanism. This study has also shown that the level of sorption is a function of the ratio of wood surface area/solution volume.     

华北典型植被根系抗拉力学特性及其与主要化学成分关系

为了解植被根系化学成分对根系力学性能的影响,该研究对华北地区常见的2种针叶树油松（Pinus tabulaeformis Carr.）和华北落叶松（Larix principis-rupprechtii Mayr.）以及3种阔叶树白桦（Betula platyphylla Suk.）、蒙古栎（Quercus mongolicus Fisch. ex Ledeb）和榆树（Ulmus pumila Linn.）5种树直径为0.75～7.65mm,共473根系进行了单根抗拉试验,并将拉伸后的根系按7个直径级归类后测定其纤维素含量、木质素含量、半纤维素含量及综纤维素含量。结果表明,5种树根系抗拉力范围为8～954 N,抗拉强度范围为6～53 MPa。不同树种间抗拉力、抗拉强度差异显著,从大到小依次为榆树＞白桦＞蒙古栎＞油松＞华北落叶松。抗拉力随直径增大以幂函数增大;抗拉强度随直径减小以幂函数和逆函数减小。根系的极限延伸率范围为6.95%～15.50%,不同树种间差异显著。5种树7个径级根系纤维素质量分数范围为20.09%～37.67%,木质素质量分数范围为18.03%～41.67%,半纤维素质量分数范围为1.29%～14.90%,综纤维素质量分数范围为26.20%～52.09%,木质素与纤维素质量分数比值（简称木纤比）为0.53～1.81。根系抗拉力与纤维素含量、半纤维含量、综纤维素含量正相关,与木质素含量、木纤比负相关;根系抗拉强度与纤维素含量、半纤维含量、综纤维素含量负相关,与木质素含量、木纤比正相关。但对于不同的树种,显著影响抗拉力、抗拉强度的化学成分不同。因此,根系中某一化学成分含量的变化并不能完全解释根系力学性能与直径的尺寸效应,或许根系其他内在因素如显微结构等也有着重要的贡献。     

Concurrent phenomena contributing to the formation of the aroma of wine during aging in oak wood: an analytical study

Red wine was stored in different oak barrels or in stainless steel, and samples were taken for two years to determine 79 aroma compounds. Aging in oak affects 41 compounds. The type of wood affects 11 compounds. At least seven different processes seem to take place concurrently in aroma evolution, and five such processes, affecting 37 compounds, are linked to the oak cask. These are extraction from the wood, oxidation of wine alcohols and amino acids, microbiological formation of ethyl phenols, sorption processes, and condensation of acetaldehyde with polyphenols. The wood can release linear gamma- and delta-lactones, beta-damascenone, and ionones. Some compounds are released very fast from wood, which suggests they lie in the external part of the wood. Some extraction profiles are too complex to be explained by physical processes. Finally, the levels of 2,5-dimethyl-4-hydroxy-3(2H)-furanone and 2-ethyl-5-methyl-4-hydroxy-3(2H)-furanone increase even in the reference wine, which suggests the presence of a precursor.