Introduction
Leaves of higher plants are covered with a cuticle layer. Leaf wax occurs within the cuticle (cuticular wax) and on the surface of the cuticle (epicuticular wax), and consists of a variety of long-chained aliphatic compounds, pentacyclic triterpenoids, phytosterols, and other minor organic compounds (
Baker, 1982). The mixture of aliphatic constituents include hydrocarbons, alcohols, ketones, esters, aldehydes and acids, each of which comprises a large number of homologues (
Eglinton and Hamilton, 1967).
n-Alkanes, which are chemically inert, occur almost ubiquitously in soils, modern and ancient sediments, crude oil and coal (
Silliman and Schelske, 2003;
Xie et al., 2003;
Schwab and Spangenberg, 2007), and their distributions have been widely used to identify the organic sources and reconstruct the vegetation history (
Ficken et al., 2000;
Freeman and Colarusso, 2001). One of the important parameters associated with
n-alkanes is the well-established carbon preference index (CPI), which denotes the relative abundance of different compounds containing odd- and even-numbered carbon atoms (
Tissot and Welte, 1984). In organic geochemistry, CPI is used to indicate the degree of diagenesis of straight-chained geolipids, with lower values being found during strong diagenesis (
Meyers and Ishiwatari, 1995). The carbon number maximum or the dominant compound, expressed as
Cmax, is used to evaluate the biological sources. Woody plants are proposed to be featured by the presence of the dominant
C27 or
C29 whilst grassy vegetation is characterized by the occurrence of the dominant
C31 n-alkane. Another parameter is the average chain length (ACL) of
n-alkanes, which describes the average number of carbon atoms per molecule based on the abundance of the odd-carbon-numbered
n-alkanes (
Poynter and Eglinton, 1990). The distribution of ACL, believed to be related to the vegetation types, has been linked to the geographical distribution of fluvial and eolian inputs, and used to identify the source regions and the contribution of petrogenic hydrocarbons in coastal marine sediments (
Poynter and Eglinton, 1990;
Jeng, 2006).
However, alkanes do change in the carbon number distributions in geological conditions. For example, alkanes react at high temperatures such as during the combustion due to the wide fire. The most common reaction of alkanes is their combustion with oxygen to produce CO
2 and H
2O. Long-chained alkanes can be chemically ‘cracked’ into smaller alkanes and alkenes by pyrolysis, and can be halogenated with elemental halogens in the presence of light (
Heider et al., 1999).
Furthermore, alkanes are biodegradable at different temperatures (
Whyte et al., 1999;
Feitkenhauer et al., 2003). A variety of enzymes efficiently and selectively catalyze the alkane oxidation at physiological temperatures and pressures (
Labinger and Bercaw, 2002). Alkanes are usually activated by terminal oxidation to the corresponding primary alcohol (
van Beilen et al., 2003). Anaerobic mineralization of alkanes involves an oxygen-independent oxidation to fatty acids (
Heider et al., 1999).
n-Alkanes are preferentially degraded over branched alkanes by most alkane-degrading bacteria (
Pirnik et al., 1974). Many microbial genera are able to grow on alkanes (
Maeng et al., 1996;
Labinger and Bercaw, 2002;
van Beilen et al., 2003). Alkane-degrading microbes were investigated in the oil industry and during the treatment of environmental pollution (
Kazunari et al., 2003;
Kato et al., 2001).
Although anaerobic and aerobic degradation of alkanes have been well studied (
Heider et al., 1999;
van Beilen et al., 2003;
van Beilen and Funhoff, 2007), it is poorly understood of the
n-alkane variation during the sinking process in the water column. Most
n-alkanes from higher plants will pass through the water column before the final burial on marine or lacustrine sediments. It is thus necessary to evaluate the variation of leaf
n-alkanes during the sinking process within the water column. We thus make a survey on the variation of leaf
n-alkanes in epicuticular waxes in natural water to evaluate the contribution of the short-time biodegradation.
Materials and methods
Sampling
Mature leaves of higher plants belonging to Setaria viridis Beauv, Cryptocarya chinensis Hemsl, Musa basjoo Sieb, Miscanthus floridulus Warb, Pteris multifida Poir. and Castanea seguinii Dode were collected in the blooming stage of the vegetation. S. viridis, M. floridulus, P. multifida and C. seguinii were abtained from Yujia hill, Wuhan (30°31′N, 114°27′E, 40 m above the sea level). Yujia hill is situated in sub-tropical zone in central China. C. chinensis and M. basjoo were obtained from Jianfengling National Nature Reserve, at the southwest of Hainan Island (18°23′-18°52′ N, 108°46′-109°02′ E, 1000-1200 m above the sea level) in south China. Jianfengling Nature Reserve is situated in the tropical zone.
Six specimens of the above plants were collected in April, 2006. The leaves were air-dried to avoid further biosynthesis. The surfaces of the leaves were washed with distilled water to remove contaminants. A part of the washed leaves were dried at 45°C in a desiccator and crushed to powder samples. The rest leaves were submerged into the tap water and decomposed at room temperature for two years, then the remains were filtrated and dried at 45°C and crushed. The powdered materials in paper bags were sealed and stored at -20°C before any further solvent extraction.
Extraction and separation of lipids
As much as 0.5 g of finely powdered air-dried leaves were extracted for 3×15 min with dichloromethane/methanol (DCM/MeOH, 93:7, v/v) in an ultrasonicator. Aliphatic fractions containing n-alkane homologues were then gained by using silica-gel column chromatography eluting with hexane. The separation and identification of the aliphatic compounds of interest was achieved for all samples by using gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS).
Gas Chromatography and GC/MS analyses
The GC analysis was conducted by using a Shimadzu 2010 gas chromatograph equipped with a ZB-1 fused silica capillary column (60 m × 0.25 mm i.d.; 0.25 μm film thickness), and a flame ionization detector (FID). Hydrogen was used as a carrier gas. The oven temperature was programmed from 70°C to 300°C at 3 °C/min, and held for 25 min at 300°C.
GC-MS analyses were performed with a Hewlett-Packard 5973A MS, interfaced directly with a Hewlett-Packard 6890 GC equipped with a DB-5 capillary column (60 m × 0.25 mm i.d.; 0.25 μm film thickness). The operating conditions were as follows: temperature ramped from 70°C to 300°C at 3°C/min, finally held at 300°C for 25 min. Helium was used as a carrier gas; the ionization energy of the mass spectrometer was set at 70 eV; the scan range was from 50 to 550 amu. n-Alkane fractions were identified by matching the retention time and the mass spectra of the alkane standards in our lab or in literature. The concentrations (from which ratios were calculated) were determined by the peak area of the compounds in ion chromatograms by referencing to the internal standard, cholestane.
Results and discussion
Table 1 provides some parameters related to
n-alkane distributions for the plant leaves and the leaf residues soaked in tap water. All the samples analyzed herein are characterized by the dominance of
C25-
C31 n-alkanes with odd-over even-carbon-number predominance (Fig. 1). The carbon number maximum (
Cmax) are
C27, C29 and
C31. Previously,
n-alkane distributions were proposed to be related to the changes of vegetation types, with dominnat
C27 or
C29 being found at woody plants and
C31 at herbaceous plants. As such, the dominant
n-alkanes extratced in sediments were used to explore the changes of plants that have populated at the watershed (
Schwark et al., 2002;
Meyers, 2003). Most of our plant lipid data appear in line with this general molecular trend but with some exceptional cases. For example,
C. seguinii is a woody plant with dominant carbon number of
C31 whilst the
Cmax of
M. basjoo, which is a herbaceous plant, is
C29. This suggests that caution should be taken into account while the vegetation is reconstructed solely on the basis of
n-alkane distributions.
It is notable that the Cmax keeps unchanged while the plant leaves were soaked in tap water for two years. The abundance of the Cmax relative to the total abundance of the odd-numbered alkanes is also found to be generally stable in most plants. However, the woody plants analyzed herein, Castanea seguinii and Cryptocarya chinensis and one of the four herbaceous plants, Setaria viridis, show an apparent increase in the abundance of the Cmax relative to the total odd-numbered n-alkanes after soaked in water. This variation might be related to the decrease in the relative abundance of either the lower-molecular-weight n-alkanes such as C23, C25 homologues in Castanea seguinii and Setaria viridis, or the heavy-molecular-weight n-alkanes such as C31, C33 homologues in Cryptocarya chinensis. The different decay processes in different plant leaves, the woody plants in particular, might cause this differentiated loss of n-alkanes.
The average chain length (ACL) is an important parameter to show the
n-alkane distributions, and the vegetation types are proposed to be the main influence on the ACL of terrigenous leaf lipids. Leaf lipids derived from grasslands may on average have longer chain length than those from woody plants (
Cranwell, 1973). In general, our data show that the four grassy plants have a greater ACL value than the two woody plants, with an exception being found at herbaceous
Musa basjoo showing an ACL value approaching the woody plants. Significantly, the ACL values keep stable after immerged into the water for two years. However, the woody plants and one of the four herbaceous plants,
Setaria viridis, are found to show a relatively larger variation in ACL values. Here again, the woody plants appear to show a large change in
n-alkane distributions after soaked in water in comparison with the herbaceous plants.
All plant species show a strong odd-carbon-numbered predominance with the carbon preference index (CPI) greater than 5. It is notable that the CPI values show a large variation after soaked in water, in striking contrast with the ACL data. In general, the herbaceous plants are observed to show a relatively small variation in CPI. The woody plants, however, show an increase in CPI values 4 times after soaked in water. Consistent with the ACL, the CPI values also show a large variation in woody plants rather than in herbaceous plants. This might arise from the different leaf texture between woody and herbaceous plant leaves, with the woody leaves being easily decomposed in aquatic environments.
It is surprising to note that the CPI values generally increase after soaked in water. It is proposed that the CPI values of leaf
n-alkanes will decrease during sinking and burial processes in aquatic environments. Bacterial reworking of hydrocarbons in both sediments and water column results in no odd-to-even carbon preference (
Grimalt et al., 1987;
Ramos et al., 1989), and thus a declined CPI value. Our data are clearly contradictory with these findings. The increase in CPI values of
n-alkanes may be related to the lower amount of microorganisms in the tap water, as well as the nature of the leaves.
The most significant property of
n-alkanes with respect to their utilization as metabolic substrates is their extremely limited solubility in water. Three possible pathways were proposed for biological uptake of hydrocarbons, i.e., soluble materials, microdroplets and macrodroplets (
Miller and Bartha, 1989). When a molecular substance dissolves in water and the new bonds are formed between them, enough energy will be released. In the case of the alkanes, the only affinity between the alkane and water molecules is from Van der Waals attraction. Terminal methyl of even-carbon-numbered alkanes is in the opposite position, much closer than those in odd-carbon-numbered alkanes. The even-carbon-numbered alkanes will have more diffuse forces. Stronger inter-molecular Van der Waals attraction gives rise to the enhanced solubility of even-carbon-numbered alkanes when present in water and thus promotes the elevated CPI values.
However, the heavier compounds exhibit both negligible solubility and slow dissolution, and the uptake of long chain alkanes by organisms following their dissolution in water is difficult to proceed. Our data presented here may also support the second and/or the third pathway. Biological uptake of hydrocarbons in droplet form is very common and frequently involves the production of biological surfactant molecules as emulsifying agents to produce microdroplets of hydrocarbons. Many hydrocarbon-degrading microorganisms have highly hydrophobic cell surfaces and may frequently associate with hydrocarbon droplets or pass into the organic phase during growth (
Watkinson and Morgan, 1990). The microbial diversity and the nature of the leaves, such as the surface textures, determine the diversity of the level of
n-alkanes decomposition.
Conclusions
The leaf n-alkanes keep unchanged in the dominant homologues when soaked in water for two years. The most significant change was observed in CPI, with enhanced values being found in leaf residues collected from water. This is contradictory with the previous reports showing the lower CPI values during sinking and burial processes in natural aquatic environments. The elevated CPI values from leaf residues might be related to the low amount of microorganisms in the water used in the simulation experiment, and the enhanced solubility of even-carbon-numbered n-alkanes via Van der Waals attraction. In contrast with herbaceous plants, woody plants show relatively greater variations in CPI and ACL values of n-alkanes. Our data show the woody plant leaves suffer from decomposition much more easily than the herbaceous leaves in aquatic environments.
Higher Education Press and Springer-Verlag Berlin Heidelberg
PDF
(144KB)
