Terpene Degradation Research Articles

Microbial degradation of plant toxins.

Plants produce a variety of secondary metabolites in response to biotic and abiotic stresses. Although they have many functions, a subclass of toxic secondary metabolites mainly serve plants as deterring agents against herbivores, insects, or pathogens. Microorganisms present in divergent ecological niches, such as soil, water, or insect and rumen gut systems have been found capable of detoxifying these metabolites. As a result of detoxification, microbes gain growth nutrients and benefit their herbivory host via detoxifying symbiosis. Here, we review current knowledge on microbial degradation of toxic alkaloids, glucosinolates, terpenes, and polyphenols with an emphasis on the genes and enzymes involved in breakdown pathways. We highlight that the insect-associated microbes might find application in biotechnology and become targets for an alternative microbial pest control strategy.

The volatile organic compounds and palatability of mixed ensilage of marigold (Tagetes erecta L.) crop residues

With increasing acreage of cash crops, the use of their by-products as supplements for livestock feed becomes an important factor. Marigold (Tagetes erecta L.) account for more than half of the world’s loose flower production. However, there is no precedent for the abundantly available marigold crop residue (MCR) being used as feed in agricultural production, probably because of its strong pungent taste. This study aimed to evaluate the biotransformation of the volatile organic compounds (VOCs) of MCR by mixed ensilage and assess its palatability by cattle. Caryophyllene, the most prevalent VOC in MCR, decreased by 29.11% (P < 0.05), 38.85% (P < 0.05), 37.15% (P < 0.05), and 28.36% (P < 0.05) ensilage with corn meal (CM), bran (BR), crop corn (CC), and straw (ST), respectively. The acetic acid content increased by 686.05% (P < 0.05), 1337.21% (P < 0.05), 1244.19% (P < 0.05), and 1795.34% (P < 0.05) after mixed ensilage with CM, BR, CC, and ST, respectively. The total amount of alcoholic VOCs followed an overall increasing trend during mixed storage and 10 new alcohols were obtained. Over seven days, feed intake of mixed ensilage MCR by cattle differed significantly (P < 0.05) among treatments compared with MCR and was highest in MCRCM. Combined with palatability trials, the best MCR feed intake was achieved with MCRCM. The findings shed light on how feed odor can be improved and how degradation of terpenes can be enhanced in practical applications by mixed ensilage.

Vaping Aerosols from Vitamin E Acetate and Tetrahydrocannabinol Oil: Chemistry and Composition.

The popularity of vaping cannabis products has increased sharply in recent years. In 2019, a sudden onset of electronic cigarette/vaping-associated lung injury (EVALI) was reported, leading to thousands of cases of lung illness and dozens of deaths due to the vaping of tetrahydrocannabinol (THC)-containing e-liquids that were obtained on the black market. A potential cause of EVALI has been hypothesized due to the illicit use of vitamin E acetate (VEA) in cannabis vape cartridges. However, the chemistry that modifies VEA and THC oil, to potentially produce toxic byproducts, is not well understood under different scenarios of use. In this work, we quantified carbonyls, organic acids, cannabinoids, and terpenes in the vaping aerosol of pure VEA, purified THC oil, and an equal volume mixture of VEA and THC oil at various coil temperatures (100-300 °C). It was found under the conditions of our study that degradation of VEA and cannabinoids, including Δ9-THC and cannabigerol (CBG), occurred via radical oxidation and direct thermal decomposition pathways. Evidence of terpene degradation was also observed. The bond cleavage of aliphatic side chains in both VEA and cannabinoids formed a variety of smaller carbonyls. Oxidation at the ring positions of cannabinoids formed various functionalized products. We show that THC oil has a stronger tendency to aerosolize and degrade compared to VEA at a given temperature. The addition of VEA to the e-liquid nonlinearly suppressed the formation of vape aerosol compared to THC oil. At the same time, toxic carbonyls including formaldehyde, 4-methylpentanal, glyoxal, or diacetyl and its isomers were highly enhanced in VEA e-liquid when normalized to particle mass.

Removal of geosmin and 2-methylisoborneol from aquaculture water by novel, alginate-based carriers: Performance and metagenomic analysis

Hydrophobic carriers were examined for geosmin and 2-methylisoborneol removal from water derived from an aquaculture system. A combination of adsorption and biodegradation was found to underlie the removal of the off-flavor compounds. Adsorption of these compounds by the carriers was unaffected by the presence of organic matter in the water to be treated. A model based on adsorption/desorption and first-order degradation kinetics provided an accurate prediction for experimentally determined 2-methylisoborneol removal rates. Steady removal of geosmin and 2-methylisoborneol as well as nitrate reduction were observed during long-term operation of the plug-flow reactors with water derived from an aquaculture facility. Metagenomic analysis of the microbial community on the carriers during long-term operation of the reactors revealed a predominance of denitrifying bacteria. It was found that geosmin and 2-methylisoborneol led to statistically significant changes in the abundances of 21 contigs that contained genes involved in terpene degradation. This study shows that at low ambient concentrations of geosmin and 2-methylisoborneol in nitrate and organic-rich water, such as found in aquaculture systems, their biodegradation can be accomplished by terpene-degrading denitrifiers that develop on hydrophobic carriers used for filtration of the contaminated water.

Enriching the nutritive value of marigold (Tagetes erecta L) crop residues as a ruminant feed by lactic acid bacteria\xa0during ensilage

BackgroundMarigold (Tagetes erecta L) accounts for over half of the world’s loose flower production, and marigold crop residue (MCR) are abundantly available and should be used as a forage. In this study, MCR from the last commercial flower pickings was ensilaged with lactic acid bacteria (LAB) and the shift in their volatile organic compounds (VOCs) profiles was monitored. Samples were collected at 6 different times during ensilage (3, 6, 9, 12, 15, 30 days) to determine and quantify the VOCs changes using a solid-phase microextraction (SPME) technique and gas chromatography – mass spectrometry (GC-MS).ResultsAfter 30 days, the caryophyllene and piperitone, which account for 14.7 and 12.1% of total VOCs, decreased by 32.9 and 9.6% respectively, alcohols increased from 2.8 to 8.1%, and the acetic acid content increased by 560%.ConclusionWe have confirmed LAB can degrade the content of terpenes and enhance the content of alcohols and acids in MCR, which was for the first time on terpene degradation in fodder by ensilage. These results have shed light on our understanding of how to improve fodder odor and to enhance terpene degradation by lactic acid bacteria fermentation.

Evaluation of Terpenes\u2019 Degradation Rates by Rumen Fluid of Adapted and Non-adapted Animals

The aim of the present study was to evaluate terpenes degradation rate in the rumen fluid from adapted and non-adapted animals. Four castrated healthy animals, two rams and two bucks, were used. Animals were daily orally dosed for 2 weeks with 1 g of each of the following terpenes, α-pinene, limonene and β-caryophyllene. At the end of each week, rumen fluid (RF) samples were assayed in vitro for their potential to degrade terpenes over time. For each animal, a 10 mL reaction medium (RM) at a ratio 1:9 (v/v) was prepared and a terpenes solution at a concentration of 100 μg/ml each, was added in each RM tube. Tubes were incubated at 39 °C under anaerobic conditions and their contents sampled at 0, 2, 4, 8, 21 and 24 h. RF could degrade terpenes as it was shown by the significantly (P < 0.05) higher overall degradation rates. Individual terpene degradation rates, were significantly (P < 0.05) higher in week 5 for limonene and marginally (P = 0.083) higher also in week 5 for α-pinene. In conclusion, the findings of the present preliminary study suggest that terpenes can be degraded in the rumen fluid.

The impact of ventilation during postharvest ripening on the development of flavour compounds and sensory quality of mangoes (Mangifera indica L.) cv. Kent

The postharvest ripening behaviour of mangoes (Mangifera indica L.) and particularly the development of colour, volatiles, sensory properties and texture, were investigated. Mangoes cv. Kent from Peru were arranged in a postharvest ripening chamber in two different ways enabling different ventilation of the fruits. Fruit properties were investigated in comparison to reference fruits after postharvest ripening for 78 h. Volatile compounds were analysed by HS-SPME GC–MS; an expert panel performed sensory analysis using descriptive methods. The arrangement of the mangoes significantly impacted the ripening procedures. Dense fruit arrangement induced a degradation of terpenes, a reduced formation of reaction products from the lipoxygenase pathway and less pronounced fruitiness and mango flavour. Principal component analysis based on volatile compounds and sensory properties showed a high correlation with the position in the ripening chamber. These data demonstrate the urgent need for further investigations of the postharvest ripening processes to increase mango quality.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprenes, and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here, we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a DTT:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently, the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism (ctm) in C. defragrans and are annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB, and CtmAB in Escherichia coli demonstrated that limonene dehydrogenase activity required both subunits, each carrying a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest placing in Enzyme Nomenclature as new entry EC 1.17.99.8.

Toxicant Formation in Dabbing: The Terpene Story.

Inhalable, noncombustible cannabis products are playing a central role in the expansion of the medical and recreational use of cannabis. In particular, the practice of “dabbing” with butane hash oil has emerged with great popularity in states that have legalized cannabis. Despite their growing popularity, the degradation product profiles of these new products have not been extensively investigated. The study herein focuses on the chemistry of myrcene and other common terpenes found in cannabis extracts. Methacrolein, benzene, and several other products of concern to human health were formed under the conditions that simulated real-world dabbing. The terpene degradation products observed are consistent with those reported in the atmospheric chemistry literature.

Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites.

Pseudomonas citronellolis is a Gram negative, motile gammaproteobacterium belonging to the order Pseudomonadales and the family Pseudomonadaceae. We isolated strain P3B5 from the phyllosphere of basil plants (Ocimum basilicum L.). Here we describe the physiology of this microorganism, its full genome sequence, and detailed annotation. The 6.95 Mbp genome contains 6071 predicted protein coding sequences and 96 RNA coding sequences. P. citronellolis has been the subject of many studies including the investigation of long-chain aliphatic compounds and terpene degradation. Plant leaves are covered by long-chain aliphates making up a waxy layer that is associated with the leaf cuticle. In addition, basil leaves are known to contain high amounts of terpenoid substances, hinting to a potential nutrient niche that might be exploited by P. citronellolis. Furthermore, the isolated strain exhibited resistance to several antibiotics. To evaluate the potential of this strain as source of transferable antibiotic resistance genes on raw consumed herbs we therefore investigated if those resistances are encoded on mobile genetic elements. The availability of the genome will be helpful for comparative genomics of the phylogenetically broad pseudomonads, in particular with the sequence of the P. citronellolis type strain PRJDB205 not yet publicly available. The genome is discussed with respect to a phyllosphere related lifestyle, aliphate and terpenoid degradation, and antibiotic resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s40793-016-0190-6) contains supplementary material, which is available to authorized users.

The molecular composition of Sicilian amber

In this study for the first time the bulk chemical composition of Sicilian amber (simetite) was determined using pyrolysis–gas chromatography–mass spectrometry with thermally assisted hydrolysis and methylation (Py(THM)–GC–MS). The results evidenced that the polymer fraction of Sicilian amber is based on a polylabdanoid structure with an enantio configuration, mainly including ozic acid and biformene. In addition, numerous monocyclic and bicyclic terpene degradation products could be detected. At higher retention times several characteristic compounds were found. Very low amounts of succinic acid could be revealed. The chemical data allowed to classify simetite as a Class 1c resinite and to suggest a botanical origin from Fabaceae.

Selecting microbial strains from pine tree resin: biotechnological applications from a terpene world.

Resin is a chemical and physical defensive barrier secreted by many plants, especially coniferous trees, with insecticidal and antimicrobial properties. The degradation of terpenes, the main components accounting for the toxicity of resin, is highly relevant for a vast range of biotechnological processes, including bioremediation. In the present work, we used a resin-based selective medium in order to study the resin-tolerant microbial communities associated with the galls formed by the moth Retinia resinella; as well as resin from Pinus sylvestris forests, one of the largest ecosystems on Earth and a yet-unexplored source of terpene-degrading microorganisms. The taxonomic and functional diversity of the cultivated, resin-tolerant fraction of the whole microbiota were unveiled by high-throughput sequencing, which resulted in the detection of more than 40 bacterial genera among the terpene-degrading microorganisms, and a range of genes involved in the degradation of different terpene families. We further characterized through culture-based approaches and transcriptome sequencing selected microbial strains, including Pseudomonas sp., the most abundant species in both environmental resin and R. resinella resin-rich galls, and three fungal species, and experimentally confirmed their ability to degrade resin and also other terpene-based compounds and, thus, their potential use in biotechnological applications involving terpene catabolism.

Mountain Pine Beetles Colonizing Historical and Naïve Host Trees Are Associated with a Bacterial Community Highly Enriched in Genes Contributing to Terpene Metabolism

The mountain pine beetle, Dendroctonus ponderosae, is a subcortical herbivore native to western North America that can kill healthy conifers by overcoming host tree defenses, which consist largely of high terpene concentrations. The mechanisms by which these beetles contend with toxic compounds are not well understood. Here, we explore a component of the hypothesis that beetle-associated bacterial symbionts contribute to the ability of D. ponderosae to overcome tree defenses by assisting with terpene detoxification. Such symbionts may facilitate host tree transitions during range expansions currently being driven by climate change. For example, this insect has recently breached the historical geophysical barrier of the Canadian Rocky Mountains, providing access to näive tree hosts and unprecedented connectivity to eastern forests. We use culture-independent techniques to describe the bacterial community associated with D. ponderosae beetles and their galleries from their historical host, Pinus contorta, and their more recent host, hybrid P. contorta-Pinus banksiana. We show that these communities are enriched with genes involved in terpene degradation compared with other plant biomass-processing microbial communities. These pine beetle microbial communities are dominated by members of the genera Pseudomonas, Rahnella, Serratia, and Burkholderia, and the majority of genes involved in terpene degradation belong to these genera. Our work provides the first metagenome of bacterial communities associated with a bark beetle and is consistent with a potential microbial contribution to detoxification of tree defenses needed to survive the subcortical environment.

Degradation of terpenes and terpenoids from Mediterranean rangelands by mixed rumen bacteria in vitro

This in vitro study aimed at estimating the disappearance rates of 14 terpenes and terpenoids after 24-h incubation with mixed bacteria from caprine rumens. These compounds comprised nine monoterpene hydrocarbons (δ-3-carene, p-cymene, β-myrcene, (E)- and (Z)-β-ocimene, α-phellandrene, α-terpinene, γ-terpinene and α-terpinolene), four oxygenated monoterpenes ((E)- and (Z)-linalool oxide, 4-terpinenol, α + γ terpineol) and one sesquiterpene hydrocarbon (β-cedrene). They were individually exposed to goat rumen microflora for 24 h in 70 ml culture tubes at an input level of 0.5 ml/l. Terpenoids were the least degraded, 100% of (E)-linalool oxide, 95% of (Z)-linalool oxide, 91% of 4-terpinenol and 75% of terpineol remained intact after 24-h incubation. In contrast, α-terpinolene concentration in fermentation broth extracts was below quantification limit, thus indicating an extensive, if not complete, degradation by rumen bacteria. Only 2% of the initial amounts of α-phellandrene were recovered. The other monoterpenes and β-cedrene were partly degraded, with losses ranging from 67% for δ-3-carene to 90% for (E)-β-ocimene. The corresponding rates of disappearance were between 2.67 and 4.08 μmol/ml inoculum per day.

Disappearance of nine monoterpenes exposed in vitro to the rumen microflora of dairy goats: Effects of inoculum source, redox potential, and vancomycin

This in vitro study evaluated the roles of diet type and redox potential in the degradation of linalool, (E)- and (Z)-beta-ocimene, alpha-phellandrene, (-)-beta-pinene, (-)-alpha-pinene, (+)-alpha-pinene, sabinene, and alpha-terpinene when incubated with rumen microflora, and it provided information on the time course of their disappearance. The 9 monoterpenes are found in the winter and spring diets of dairy goats in northwestern Mediterranean grazing systems. The diets were individually exposed to rumen microflora for 3 h in 17-mL culture tubes at a concentration of 4 microL/L. The mixed flora of the inoculum was controlled by the use of vancomycin (eliminating gram-positive bacteria) and by the energy source (starch vs. structural polysaccharides) on which rumen microflora had been grown. Redox potential was controlled by addition of L-cysteine-hydrochloride. The preliminary adaptation of microbial inoculum to a diet rich in structural carbohydrates reduced the recovery yields of (E)- and (Z)-beta-ocimene, (-)-beta-pinene, (-)-alpha-pinene, (+)-alpha-pinene, and sabinene (P<0.01), whereas vancomycin was without effect. The effect of carbohydrate source likely stems from the specific composition of the microbial community rather than from its acidogenic capacity. Reducing the culture redox potential by 50 mV reduced the recovery yields of linear and monocyclic terpenes (P<0.02), and the culture redox potential interacted with the inoculum source for (E)- and (Z)-beta-ocimene and for alpha-phellandrene. The time course of terpene disappearance was studied by exposing terpenes to a rumen microflora adapted either to starch or to fiber for 3, 6, or 24 h. The degraded fraction reached a plateau within 3 h for alpha-phellandrene and for all the isomers of beta-ocimene and pinene with the fiber-adapted microflora as well as with both inocula for alpha-terpinene. With the starch-adapted microflora, this steady state was reached for most other terpenes within 6 h of incubation. Sabinene was the only compound still disappearing after an incubation period of 6 h. Biotic and environmental variables in the rumen affected terpene degradation in a way that can alter the dietary terpene profile and possibly its influence on animal product characteristics.

The Role of Ozone-reactive Compounds, Terpenes, and Green Leaf Volatiles (GLVs), in the Orientation of Cotesia plutellae

The emission of inducible volatile organic compounds (VOCs), i.e., inducible terpenes, and green leaf volatiles (GLVs), is a common response of plants to herbivore attack. These VOCs are involved in the orientation of natural enemies, i.e., predators and parasitoids, toward their herbivore prey or hosts (indirect defense of plants). Terpenes and some GLVs are readily oxidized by ozone (O(3)), an important oxidant of the low atmosphere and predicted to increase as a result of anthropogenic activity. It has been recently reported that O(3) degradation of terpenes and GLVs does not affect signaling in two selected tritrophic systems. Natural enemies may have learned to use oxidation products that are more stable in nature to locate their prey. To understand the role of these compounds on the tritrophic system Brassica oleracea-Plutella xylostella-Cotesia plutellae, we assessed the preference of wasps to different combinations of cabbage VOCs (intact vs. herbivore-induced and herbivore-induced vs. herbivore-induced VOCs) in the presence or absence of O(3). We found that C. plutellae preferred P. xylostella-damaged plants at 0 and 120 nl l(-1) O(3) to intact plants at 0 nl l(-1) O(3). However, wasps preferred P. xylostella-damaged plants at 0 nl l(-1) to P. xylostella-damaged plants at 120 nl l(-1) O(3). The results suggest that compounds other than terpenes and GLVs are crucial for the orientation of the wasps, but terpenes and GLVs contribute to the behaviorally active VOC blend of herbivore-damaged cabbages by increasing their attraction to them. The products resulting from oxidation of terpenes and GLVs do not seem to play a role in the host location process as speculated previously.

Terpene degradation and extraction from basil and oregano leaves using subcritical water

In the first part of this study, the stability of five terpenes (α-pinene, limonene, camphor, citronellol, and carvacrol) under subcritical water conditions was investigated. The stability studies were carried out at four different temperatures (100, 150, 200, and 250 °C) with two different heating times (30 and 300 min). When water temperature was increased, the degradation of terpenes became more serious. Prolonged exposure time to each heating temperature also caused decreased terpene stability. The terpene recoveries were determined by conducting subcritical water extraction of sand spiked with terpenes. The recoveries are typically around 70 to 80% for extractions at 100 °C. Terpene recoveries were decreased with increasing water temperature due to poorer stability of terpenes. After the degradation and recovery studies, basil and oregano leaves were extracted using water at both 100 and 150 °C. The concentrations of each individual terpene in the water extract generally ranged from trace quantity to 65 μg terpene/g herb. However, the concentration of carvacrol in the oregano-water extract at 150 °C was found to be as high as 4270 μg carvacrol/g oregano.

Emission fluxes and atmospheric degradation of monoterpenes above a boreal forest: field measurements and modelling

The contribution of monoterpenes to aerosol formation processes within and above forests is not well understood. This is also true for the particle formation events observed during the BIOFOR campaigns in Hyytiälä, Finland. Therefore, the diurnal variation of the concentrations of several biogenic volatile organic compounds (BVOCs) and selected oxidation products in the gas and particle phase were measured on selected days during the campaigns in Hyytiälä, Finland. α-pinene and Δ3-carene were found to represent the most important monoterpenes above the boreal forest. A clear vertical gradient of their concentrations was observed together with a change of the relative monoterpene composition with height. Based on concentration profile measurements of monoterpenes, their fluxes above the forest canopy were calculated using the gradient approach. Most of the time, the BVOC fluxes show a clear diurnal variation with a maximum around noon. The highest fluxes were observed for α-pinene with values up to 20 ng m−2 s−1 in summer time and almost 100 ng m−2 s−1 during the spring campaign. Furthermore, the main oxidation products from α-pinene, pinonaldehyde, and from β-pinene, nopinone, were detected in the atmosphere above the forest. In addition to these more volatile oxidation products, pinic and pinonic acid were identified in the particle phase in a concentration range between 1 and 4 ng m−3. Beside these direct measurement of known oxidation products, the chemical sink term in the flux calculations was used to estimate the amount of product formation of the major terpenes (α-pinene, β-pinene, Δ3-carene). A production rate of very low volatile oxidation products (e.g., multifunctional carboxylic) from ·OH- and O3-reaction of monoterpenes of about 1.3·104 molecules cm−3 s−1 was estimated for daylight conditions during summer time. Additionally, model calculations with the one-dimensional multilayer model CACHE were carried out to investigate the diurnal course of BVOC fluxes and chemical degradation of terpenes.

Thermal Degradation of Terpenes: Camphene, Δ3-Carene, Limonene, and α-Terpinene

Emissions from wood dryers have been of some concern for a number of years, and recent policy changes by the Environmental Protection Agency have placed emphasis upon the gaseous emissions that lead to the formation of particulate matter as small as 2.5 μm diameter. In this qualitative study, camphene, Δ3-carene, limonene, and α-terpinene were thermally degraded in the presence of air to determine the number and kind of oxidative degradation products that might be expected under drying conditions used in processing wood products. Various chromatographic methods were used to isolate the products for proof of structure by NMR and/or GC−MS. The degradation products resulted from dehydrogenations, epoxidations, double bond cleavages, allylic oxidations, and rearrangements. A number of compounds not previously associated with the thermal degradation of these terpenes were identified.

Long Term Degradation of Terpenes. From a 20 Million Years Old Taxodioxylon gypsaceum

The comparison of the steam distillate from a 20 million years old Taxodioxylon gypsaceum with that of a corresponding 1.5 million years old sample has permitted the description of the last steps of terpene degradation of this species. The main feature is the reduced number of components in the older sample which contains over 97% of α-phyllocladane and traces of other components present, by contrast, in the younger one. The lack of new compounds, related to the lost components, suggests leaching to be the cause of the losses. This seems to be confirmed by the analysis of the inner part of a trunk of one of the younger samples presently subjected to a sort of natural leaching. Analyses indicate an increase in α-phyllocladane and a severe reduction in simonellite as well as other terpenes, together with the formation of two new compounds of 164 and 222MWs. The occurrence of α-phyllocladane in other European lignites suggests the choice of this compound as a biomarker of the species Taxodioxylon gypsaceum.