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Decomposition process in the Mediterranean region. Chemical compounds and essential oil degradation from Myrtus communis
International Biodeterioration & Biodegradation 64 (2010) 356e362
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Decomposition process in the Mediterranean region. Chemical compounds and essential oil degradation from Myrtus communis Christos N. Hassiotis a, *, Diamanto M. Lazari b a b
Higher Technical University of Larissa, Department Natural Environment and Forestry, 43100 Karditsa, Greece Aristotle University of Thessaloniki, School of Pharmacy, Laboratory of Pharmacognosy, 54124, Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2010 Received in revised form 31 January 2010 Accepted 2 February 2010 Available online 24 April 2010
All aromatic plants emit volatile substances into the environment either during life cycle or during decomposition. These volatile constituents affect the top soil microflora, the nutrients recycle process and the vegetation establishment. Myrtus communis is a perennial aromatic shrub, rich in aromatic substances which can be found abundant in the Mediterranean. Fresh mature leaves of myrtle were used for this study using the litterbag technique. The essential oil content of the initial plant was 0.62% dry weight (dw) and after eighteen months burring dropped to 0.05%. The major oil compounds were 1,8-cineole (29.6%), a-pinene (24.7%) and myrtenyl acetate (10.6%). The essential oil degradation rates were similar under the relative small area of the investigation. Terpenes, esters and alcohols were released fast from buried material. The bacterial activity was induced by the presence of myrtle volatile oil. The only compounds that remained after eighteen months were 1,8-cineole and camphene. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Myrtus communis Essential oil Decomposition Bacteria Biorementation
1. Introduction Aromatic plants which contain essential oils are one of the major plant categories in the Mediterranean region. These plants have the capacity to synthesize, accumulate and emit volatiles that may act as aroma and flavor molecules due to interactions with living organisms. These low-molecular-weight substances derived from the fatty acid, amino acid and carbohydrate pools constitute a heterogenous group of molecules and cyclic structures bearing various functional groups (e.g. alcohols, aldehydes, ketones, esters and ethers) and also nitrogen and sulfur (Schwab et al., 2008). Almost all plants, and mainly the aromatic ones, emit volatile substances (Yang et al., 2009). The essential oil into aromatic plants represents 0.1e3% of the dry weight, and there are several ways that these secondary metabolites escape into the environment. Essential oils do not survive forever in plant material and the fade of the oil following leaf fall is a topic that requires more research. According to Margaris and Vokou (1986) the terpenoid emissions participate in photochemical reactions leading to aerosol production. Essential oil decomposition can occur even when the plant is placed in the dark. As Sombrero (1992) stated, the longer the plant remained in the dark the higher the drop in oil content, reaching approximately * Corresponding author. Tel.: þ30 6946 501110; fax: þ30 23920 92221. E-mail addresses: [email protected] (C.N. Hassiotis), [email protected] (D.M. Lazari). 0964-8305/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2010.02.007
40% in a period of twenty-four days. Plant essential oils are incorporated in plant material and follow the litter fall. Litter deposit depends primarily on the productivity of plant communities, which in turn are affected by climate, soil fertility, soil water retention and species composition (Pausas, 1997). The decomposition of the essential oil is achieved by the presence of microorganisms which need to have the enzymatic capacity to break down the organic compounds of the litter. The litter and its components constitute source of carbon and nutrients (Melillo and Aber, 1984). Plant essential oils constituents are insoluble or almost insoluble in water. The terpenoids which are characterized by their liability (Knobloch et al., 1989) have been found to interfere with enzymatic reactions of energy metabolism. The presence of essential oils or individual compounds derived from essential oils in the top soil layer has been found to enhance biodegradation (Tandlich et al., 2001; Isidorov and Jdanova, 2002; Rhodes et al., 2007; McLoughlin et al., 2009; Suttinun et al., 2009). The most prominent among the organisms known to attack hydrophobic residues are members of the genus Pseudomonas and Nocardia (Gunsalus and Marshall, 1974; Zorn et al., 2004; Solyanikova et al., 2008; Marostica and Pastore, 2009). It was also found (Stevenson, 1967) that Arthrobacter spp. are able to utilize a great number of aromatic hydrocarbons as their sole carbon source. There are a number of reports dealing with the ability of Arthrobacter in degrading aromatic structures (Shimoni et al., 2003). Gibbon and Pirt (1971) found that the volatile oil is decomposable by bacteria and that at least six species of Pseudomonas were able to degrade
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a-pinene, a common constituent of volatile oils. Kaplan and Hartenstein (1979) and Harder and Foß (1999) found that low concentrations of toluene in cultural media encouraged the growth of 19 species of bacteria and fungi which used toluene as their only source of carbon. On the other hand 1,8 cineole inhibits respiration (Muller et al., 1969) and alters the anatomy of the roots and seedling cells. As Halligan (1975) reported, camphor and 1,8-cineole were the two most toxic components and contribute to toxicity in the field. In the literature there are many reports for essential oil biosynthesis, essential oil composition and toxicity, biorementation using plant essential oils, for litter decomposition and the nutrient feedback (Halligan, 1975; Harder and Foß, 1999; Rhodes et al., 2007; Suttinun et al., 2009; Wannes et al., 2009). Despite on going research the role of essential oil degradation in plant litter in situ is ignored by many scientists. The rate of essential oil disappearing determines the remaining oil in plant litter especially when litter is accumulated year after year resulting in higher flammability, in microorganisms inhibition or activation, in nutrients immobilization, in allelopathic effects on other plants growth. The aim of this study was to investigate the degradation of essential oil during the decomposition process of myrtle (Myrtus communis), the way that the major compounds are released during that process and how bacteria populations are influenced by the presence of these compounds. 2. Materials and methods 2.1. Experimental area The area of the investigation was in the North part of Greece at Chalkidiki peninsula (latitude 40180 N; longitude 23 320 E). The peninsula and the experimental area is typically Mediterranean, characterized by hot dry summers and cool winters where the lower temperatures do not exceed five degrees below zero. The area of the investigation was rich in aromatic plant species. M. communis which was found in transitional vegetation communities was used for essential oil evaluation during decomposition process. 2.2. Experiment installation The litterbag technique (Alhamd et al., 2004) was used to evaluate the litter decay, the fade of the essential oil content and its chemical compounds disappearing. Freshly abscised leaves of M. communis were collected in the study area (8000 m2) by shaking 20 Myrtle shrubs over a large net at the end of July where the essential oil content is quite high. Any debris was carefully removed from each collection. The raw material was mixed, the essential oil was extracted and measured and the oil composition was evaluated by GCeMS analysis. Four kilos of raw material were used and the samples were put in litterbags. Leaf samples (about 20 g fw) were enclosed in terylene net bags (16 10 cm). The mesh size (1 mm2) was small enough to prevent the loss of leaf fragments, but large enough to allow aerobic microbial activity and the entry of small soil animals. Twelve replicates, of sixteen bags, in total 192 litterbags, were installed in the experimental area under twelve myrtle shrubs which were selected randomly, on the 1st of August 2006. The experiment was carried out for 18 months which including one month of preparations. 2.3. Sample collection The first sampling was on the 1st of September 2006 (twelve litterbags, one of each replication) and then periodically, every month. The extraction of the oil was achieved by organic solvent (Fig. 1). In the solvent extraction the plant material was put into a plastic pipette tip and was kept by nonabsorbent cotton from both sides. The cotton on the bottom filters the solvent and regulates the flow through the plant
Fig. 1. Schematics for essential oil solvent extraction. The plant material is trapped by nonabsorbent cotton. The macerate is washed with solvent (diethyl ether) in order to extract the oil.
material thus controlling the time of extraction. The cotton on the top prevents accidental losses of the plant material when applying the solvent. The extraction medium was diethyl ether with 104 M (M) of n-tetradecane to act as an internal standard for any losses (Hassiotis, 1997). Approximately 2 g of myrtle was put into the capsule after a precise weighting and covered by the top cotton. 5 ml of solvent (diethyl ether) was added on the top and left to flow through the plant sample, percolating the essential oil. The washing lasts about 15 min and three successive washings were taken. At the end of each washing an elastic tube was fitted on the open top, which was connected with an air pump. The air pressure breaks the structures which are resistant to the solvent treatment. The dilution was transferred and left at room temperature for evaporation (in a fume cupboard) and the essential oil was measured. Three samples of each litterbag were solvent extracted for precise essential oil evaluation. Moreover, 2 g from the litterbags were used to calculate the humidity of the samples and to express the oil content per dry weight. 2.4. Essential oil analysis The composition of the volatile constituents was established by GCeMS analysis. GCeMS analysis were performed on a Shimadzu GC-2010 e GCMS-QP2010 system operating in EI mode (70 eV) equipped with a split/splitless injector (230 C), a split ratio 1/30, using a fused silica HP-5MS capillary column (30 m 0.25 mm (i.d.), film thickness: 0.25 mm). The temperature program was from 50 C (5 min) to 290 C at a rate of 4 C min1. Helium was used as a carrier gas at a flow rate of 1.0 mL min1. Injection volume of each sample was 1 mL. Retention indices for all compounds were determined according to the Van den Dool approach (Van den Dool and Kratz, 1963), using n-alkanes as standards. The identification of the components was based on comparison of their mass spectra with those of NIST21 and NIST107 (Massada, 1976) and those described by Adams (2001) as well as by comparison of their retention indices with literature data (Bisio et al., 1998; Davies, 1990). All data have been tested for statistically important differences with analysis of variance test using SPSS 16.0 for windows. 2.5. Bacteria analysis Three samples of 1 g per litterbag were collected, cut into small pieces (approximately 5 mm) and dissolved into 100 ml Ringers
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solution (500 ml distilled water plus one tablet for stock Ringers solution). After 5 min in suspension, one ml of each solution was transferred into a further 100 ml Ringers solution and a serial dilution of one more step carried out. After the dilution the total concentration was equivalent to 1g 106 of myrtle litter per ml Ringers solution. Agar substrate was prepared a day before using agar powder, bacteriological peptone, and yeast extract in the ratio 1.5%, 1%, 1% respectively. Distilled water was used for the dilution. In order to avoid any contamination the agar was sterilized in an autoclave at 120 C and 2 atm pressure for 30 min. The petri-dishes (8 cm in diameter) were filled with this agar substrate which was left for 24 h period to check for contamination. After checking, the clear petri-dishes were used for bacteria cultivation. One ml was pipetted from the final Ringers dilution (106) and was spread on the agar surface (three petri dishes were used for each sample). A sterilized glass spreader was used for the drop spreading and then rotated clockwise and anticlockwise to get homogenous distribution of the suspension to the medium. The plates were marked and incubated in a sterilized chamber, at 26 C. The number of bacterial colonies was counted 48 h, after spreading and recorded following the colony forming unit (CFU/g) standards. The bacterial colonies were counted from nine petri dishes of each litterbag, analyzed by using StatistiXL Version 1.8 add-in for MS Excel (Nedlands, Western Australia) and presented as mean standard deviation. 3. Results and discussion 3.1. Bacterial colonies The mean number of bacterial colonies developed on agar medium 48h after spreading is presented in Fig. 2. At the date of experiment installation on the 1st of August the average bacterial colonies in myrtle leaf material was 198.1 106 g1. After one month the bacterial colonies in burring myrtle increased 27.7% and after two months reached up to 288.2 106 g1 in October and 286.4 106 g1 in November. This period apart from fresh myrtle “food” for bacteria coincides with favorable weather conditions. The bacterial activity started to decline from December to August, where the minimum activity was recorded. On the 1st of August 2007, the bacteria numbers dropped at 103.1 106 g1 and then slightly increased up to
300
1.00
200 0.60
6
essential oil (%) dry weight
0.70
-1
250 0.80
bacterial colonies x 10 g dry weight
0.90
150
0.50 0.40
100 0.30 0.20 50
Oil content 0.10
0 Dec-07
Oct-07
Nov-07
Sep-07
Jul-07
Aug-07
Jun-07
Apr-07
May-07
Mar-07
Jan-07
Feb-07
Dec-06
Oct-06
Sep-06
Aug-06
0.00
Nov-06
Bacterial colonies
Fig. 2. Essential oil percentage derived from buried M. communis (left y axis). The hi-low lines present oil fluctuation between replications. Right y axis: Bacterial colonies (standard deviation) developed from litter of M. communis. The plant material was buried from August 2006 to December 2007.
the end of the study, reaching at 122.4 106 g1. Comparing the period of October and November 2006 with the respective period of October and November 2007 (with similar weather conditions) it is revealed 138% higher bacterial activity in the first period. This observation leads us to the conclusion that fresh buried myrtle material (two months old) was used as carbon source and enhanced bacterial activity. 3.2. Essential oil decomposition The essential oil content obtained in our study is presented in Fig. 2. During the period of the investigation the essential oil content of buried Myrtus communis presented a remarkable decrease. After a period of eighteen months the essential oil content was reduced at about 93% (from 0.62% to 0.05%). This means that almost the total amount of myrtle oil has been disappeared during the period of eighteen months. Even after this great oil losses, a small amount of oil remained into plant material at the end of the study. Analysis of variance which was performed in order to test the remaining essential oil into plant material during the study resulted in significant differences between the dates of samplings (P < 0.01). In other words, in every new monthly sampling there were remarkable losses of essential oil from buried myrtle. The differences in essential oil content between experimental sites, for the same dates of samplings, were also tested by ANOVA and they did not present any differences leading to the conclusion that in a small area, as the one of this study, the essential oil degradation rates were presented as more or less uniform. The aim of the twelve replications of litterbags was targeted to ensure precise results in oil disappearance and not to investigate the influence of locality in decomposition rates. While the experiment installation was in August the initial oil losses recorded in October and up to December. From January to March there is a slow down whereas from April to June there is an increase in the rhythm that the oil disappeared. The same pattern observed from July to September and then increased again up to the end of the year (Fig. 2). There were differences in the two periods of the study, October to December 2006 and January to March 2007. In the first period a remarkable oil disappearance is shown, which is possibly correlated with the start of rainfalls in the autumn and higher bacterial activity, whereas in the second group, even with higher precipitations, the oil losses are rather low. This observation supports the opinion that the most important, among environmental parameters affecting decomposition in Mediterranean, is the temperature during summer and winter. Even though the higher precipitations in January, February and March the oil losses are restricted due to the low temperatures which in turn affect the bacterial and microbial activity. Berg et al. (1982) reported that the constituents of the litter can be divided into three broad groups which begin their net mass loss at different stages of decomposition. Concerning the essential oil degradation or disappearance, Knobloch et al. (1989) and later Rajeswara Rao et al. (2006) reported that the slight water solubility of terpenes would allow some leaching loss of these components and moreover the biological effect of the oil constituents is related to their solubility in water and/or their solubility in membrane lipids. The solubility of terpenes, in addition to favorable climatic conditions for microbial activity is possibly the reason why a remarkable oil loss is presented from October to December. The present results do not indicate that losses are due to evaporation. At the date of experiment installation and for a month period (August to September 2006) where the oil content was higher in myrtle and the temperatures very high (day average 26.7 C) did not record oil disappearance. It is indicated from this study that the essential oil disappearance is depending from bacterial activity which in turn is limited by environmental factors such as temperature and rainfalls.
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The microorganisms during summer are limited by high temperatures and during winter by low. The average temperature of the coldest month is 5.6 C, whereas that of the hottest month is 26.7 C. August is the driest month of the year with an average rainfall of 14.3 mm (data of the meteorological station of Aristotle University of Thessaloniki, the nearest to the study area). Oil leaching and possibly immediate consumption by bacteria is recorded only during the first wet period after installation whilst the disappearance through evaporation is almost zero. The knowledge of essential oil amount into buried plant material has one more ecological aspect because essential oils have also major roles in presence of other organisms apart from microorganisms. There are several reports on the effect of essential oils and its chemical compounds on other organisms. Singh et al. (2002) working with oil from Coleus amboinicus found it to be insecticidal to white termites (Odontotermes obesus Rhamb.) with 100% mortality at a dose of 2.5 102 mg cm3 for 5 h exposure. Actually the oil from C. amboinicus was also more active than the synthetic insecticides, Thiodan and Primoban-20, against termites. Kanat and Alma (2004) indicated that the essential oils from nine plant species and sulfate turpentine were effective against the larvae of Thaumetopoea pityocampa. Panella et al. (2005) reported the ability of essential oil components to kill arthropods at relatively low concentrations represents an alternative to the use of synthetic pesticides for control of disease vectors. 3.3. Essential oil analysis The essential oil analysis demonstrated that forty seven compounds comprise 98.8% of the total oil. Seventeen compounds presented percentage more than 0.1% w/w (Table 1) comprising the 94.80% of the total oil. The two major compounds were 1,8-cineole and a-pinene with an average percentage of 29.60% and 24.71% respectively. Other predominant compounds were myrtenyl acetate (10.60%), limonene (6.91%), a-terpineol (6.10%) and linalool (4.30%). The results of essential oil analysis of this study for myrtle in Greece are in agreement with relative work done recently in the Mediterranean region (Messaoud et al., 2005; Wannes et al., 2007; Pereira et al., 2009 and Wannes et al., 2009) as well as around the world (Ashnagar et al., 2009; Pezhmanmehr et al., 2009). Four groups of compounds participate in the chemical composition of Table 1 Chemical composition of Myrtus communis essential oil used for the experiment. Compoundsa
RIb
Composition (%) SDc
a-Pinene
932 946 974 1028 1029 1100 1190 1197 1228 1256 1257 1324 1351 1355 1366 1385 1402
24.71 0.81 2.02 6.91 29.60 4.30 6.10 0.99 1.16 0.80 1.49 10.60 0.99 0.87 1.13 0.40 1.92 94.80
Camphene b-Pinene Limonene 1,8-Cineole Linalool a-Terpineol Myrtenol Nerol Geraniol Linalyl acetate Myrtenyl acetate a-Terpinyl acetate Eugenol Neryl acetate Geranyl acetate Methyl eugenol TOTAL
0.76 0.02 0.04 0.34 1.19 0.25 0.33 0.04 0.08 0.02 0.04 0.41 0.04 0.03 0.03 0.04 0.06
a Only compounds contributing more than 0.1% of the total oil are presented. Compounds listed in order of elution from an HP-5MS column. b Retention indices as determined on an HP-5MS column using a homologous series of n-alkanes. c Standard deviation.
359
myrtle oil. These are terpenes (34.4%), esters (14.6%), alcohols (16.2%) and ethers (29.6%). The way that the individual chemical compounds are released from buried M. communis essential oil are presented (compound average) in Table 2. All compounds during this study of eighteen months showed different decrease rates. There are remarkable losses for each compound percentage for all experimental sites. The only compounds remaining into plant material after a year period were camphene, 1,8-cineole and methyl eugenol while at the end of the study only camphene (0.1%) and 1,8-cineole (4.8%) were present in the remaining oil. We could infer that the remaining 1,8-cineole (4.8%) is due to the high initial percentage (29.6%) of this compound in the total oil although this did not also happen in a-pinene. The initial 24.7% of this compound in the total oil disappeared within a year period. This is an indication that the initial high percentage of a compound in an essential oil cannot be a reliable index for the presence of this compound in plant litter after a half or a year period and generalizations cannot be done. In a previous study, Hassiotis (1997) working with Laurus nobilis essential oil degradation and compounds release found that after fifteen months period the only compounds remained in the final essential oil were 1,8 cineole and camphor. Most of the compounds disappeared easily from the plant litter mainly by microorganism consumption and less by leaching or evaporation. The importance of microorganisms to decompose natural aromatic structures has been reported by Harder and Foß (1999). In 1988, Vokou and Margaris found that under favorable climatic conditions (sufficient moisture) soil microorganisms have the capacity to decompose natural products, such as secondary metabolites, at a rate of at least 1.7 g m2 d1. Even 1,8-cineole, which is reported as very toxic to microorganisms (Kivanc and Akgul, 1986) can be degraded by microorganisms. This cineole toxicity possibly resulted to be present in the final plant litter after eighteen months of degradation. The way that the groups of compounds are released during decomposition is presented in Fig. 3 with intervals of four months. There is a remarkable decrease in groups of terpenes, alcohols and esters whereas ethers (mainly 1,8 cineole) degradation rates are slow. The total amount of terpenes, alcohols and esters in the initial material was 65.2%, in December 2006, 28.9%, in April 2007, 7.0% and in August 2007 only 0.1%. The fast degradation of these groups of compounds is positively correlated with the high bacterial activity in the first eight months of the experiment. The correlation between fast (terpenes, alcohols and esters) and slow (ethers) decomposable groups of compounds in myrtle oil and bacterial colonies developed from myrtle litter are shown in Fig. 4. It is observed a two way relation between rapid released groups of compounds and bacterial activity. The fast release of the compounds is due to bacterial activity or the higher bacterial activity is induced by the presence of easily decomposable compounds. The high bacterial activity seems not to influence the degradation of ethers (mainly 1,8-cineole) whose rate is slow. 1,8-cineole is known as compound causes bacteria inhibition. This inhibition did not manifest up to January 2007 where equilibrium between faster and slower degraded compounds were observed. From March to August 2007, where the easily decomposable compounds have almost disappeared, the pattern of bacteria follows the pattern of ethers. It is worth noting that for that period as the temperature increased rainfall decreased. The rapid release of terpenes has an ecological approach since they induce degrading bacteria. There is speculation that volatile organic compounds (VOCs) emitted within soil either by roots or by decaying biomass may enhance the biodegradation of persistent organic pollutants through cometabolism. Several studies have demonstrated the role of monoterpene compounds in the induction or enhancement of polychlorinated biphenyl (PCB) biodegradation. These many plant-derived chemicals, including also those
C.N. Hassiotis, D.M. Lazari / International Biodeterioration & Biodegradation 64 (2010) 356e362 40
groups of compounds (%)
1.1 0.9 0.7 0.5 0.3 0.2 0.1 0.1 e e e e e e e e e
0.4 0.2 0.1 0.1 0.1 e e e e e e e e e e e e
1.9 1.6 1.5 1.5 1.5 1.4 1.3 1.1 0.9 0.8 0.8 0.6 0.4 0.3 0.3 e e
35 30 25
ethers terpenes
20
alcohols esters
15 10
0.9 0.9 0.9 0.7 0.6 0.5 0.4 0.3 0.2 0.1 e e e e e e e
eugenol
neryl acetate
geranyl acetate
methyl eugenol
360
0
Apr-07
Aug-07
Dec-07
Fig. 3. Groups of chemical compounds participating in chemical composition of Myrtus communis essential oil. The plant material was buried for seventeen months. The highest and the lower amount of each group are presented by Y error bars.
generated from root turnover, stimulate microorganisms to biodegrade xenobiotics (Isidorov and Jdanova, 2002). Steinbrecher et al. (1999) attempted to evaluate the terpenoid emissions working with Citrus species and found that were in the range of 1500e3000 pmol mm2 s1 from soils under the trees covered with litter, and of 10e100 pmol mm2 s1 from bare soils between rows. The origin of emissions from leaf and fruit litter was experimentally confirmed by removal of the uppermost soil layers. Focht (1995) proposed that plant terpenes might be the natural substrate for PCB oxidation, rather than biphenyl. Later Hernandez et al. (1997) demonstrated that soils enriched with orange peel, ivy leaves, pine needles or eucalyptus leaves resulted in 105 times more biphenyl utilizers (108 g1) than unamended soils (103 g1) simultaneously inducing Aroclor 1242 degradation. Gilbert and Crowley (1997) screened several terpenoid compounds for their ability to induce PCB biodegradation in Arthrobacter sp. strain B1B and resulted that s-carvone, the principal chemical component of
300
6
groups of compounds (%)
200 40 150 30 100 20
50
0 Dec-07
Oct-07
Nov-07
Sep-07
Jul-07
Aug-07
Jun-07
Apr-07
May-07
Mar-07
Jan-07
Feb-07
Oct-06
Sep-06
Dec-06
10
Aug-06
24.7 22.4 19.4 14.9 10.7 7.5 5.2 3.4 2.1 1.1 0.4 0.2 0.1 e e e e
250
50
Nov-06
0.8 0.7 0.7 0.6 0.6 0.6 0.6 0.5 0.5 0.4 0.3 0.3 0.3 0.2 0.2 0.1 0.1
2.0 1.8 1.6 1.2 0.9 0.6 0.4 0.3 0.2 0.1 e e e e e e e
6.9 7.0 6.7 5.7 4.5 3.5 2.7 2.0 1.4 0.8 0.3 0.1 0.1 e e e e
29.6 29.8 28.6 25.3 23.5 23.1 23.0 21.6 20.5 16.4 13.4 12.2 11.7 8.7 6.7 5.1 4.8
60
0
Aug-06 Sep-06 Oct-06 Nov-06 Dec-06 Jan-07 Feb-07 Mar-07 Apr-07 May-07 Jun-07 Jul-07 Aug-07 Sep-07 Oct-07 Nov-07 Dec-07
1,8-cineole limonene
b-pinene camphene
a-pinene
Compounds of Myrtus communis essential oil (%)
Terpenes & Alcohols & Esters Ethers Bacterial colonies
-1
6.1 5.8 5.3 4.3 3.3 2.4 1.8 1.2 0.8 0.4 0.2 0.1 e e e e e 4.3 3.9 3.4 2.6 1.9 1.3 0.9 0.6 0.4 0.2 0.1 e e e e e e
Dec-06
bacterial colonies x 10 g dry weight
a-terpineol linalool
1.0 1.0 0.8 0.6 0.5 0.4 0.3 0.2 0.1 e e e e e e e e
1.2 1.1 0.9 0.6 0.5 0.4 0.2 0.2 0.1 e e e e e e e e
0.8 0.8 0.7 0.5 0.4 0.3 0.2 0.2 0.1 e e e e e e e e
1.5 1.0 0.7 0.4 0.2 0.1 0.1 e e e e e e e e e e
10.6 8.1 5.8 4.1 2.8 1.9 1.2 0.6 0.2 0.1 e e e e e e e
1.0 0.7 0.5 0.3 0.2 0.1 0.1 e e e e e e e e e e
Aug-06
70
Date
Table 2 Average percentage of the main compounds found in essential oil of buried Myrtus communis.
myrtenol
nerol
geraniol
linalyl acetate
myrtenyl acetate
a-terpinyl acetate
5
Fig. 4. Release rates of terpenes, alcohols and esters in comparison with release rate of ethers derived from M. communis essential oil (left y axis). Right y axis: Bacterial colonies (standard deviation) developed from litter of M. communis. The plant material was buried from August 2006 to December 2007.
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spearmint (Mentha spicata), induced the biotransformation of 62% of Aroclor 1242, or 26 of 32 identified peaks. Tandlich et al. (2001) used carvone and limonene to stimulate biodegradation of Delor 103 (another commercial mixture of PCBs) by Pseudomonas stutzeri. Additional PCB congener removal occurred after supplementing with terpenes compared with control-grown cultures. In 2003, Oh et al. examined the ability of terpenes to prolong the survival of a known PCB-degrading bacterium, Pseudomonas pseudoalcaligenes KF707, in soil. The addition of 50 mg l1 p-cymene or 50 mg l1 a-terpinene increased KF707 survival by 10e100 fold compared with biphenyl supplemented and control mesocosms. Rhodes et al. (2007) concluded that the degradation of 2,4-dichlorophenol by indigenous microorganisms is greater in those soils amended and ‘aged’ with monoterpenes, (a-pinene, limonene and p-cymene) than in freshly spiked or control soils. She also suggested that the remediation of contaminated soils may be promoted by stimulating indigenous microorganisms either through planting monoterpeneemitting vegetation, particularly pine species, or through direct application of natural substances with high monoterpene content, for example pine needle litter or citrus waste. Lately McLoughlin et al. (2009) suggested that monoterpenes can stimulate the biodegradation of 2,4-DCP by indigenous soil microorganisms and that monoterpene amendment in soils is an effective strategy for removing organic contaminants. Therefore, it is feasible to suggest that there is potential for in situ remediation of contaminated soils through the stimulation of indigenous microorganisms through applications of exogenous isoprenoid such as terpene rich plant residues or the planting of isoprenoid-emitting vegetation. Recently Suttinun et al. (2009) working with induction of TCE (Trichloroethylene)-degrading enzymes with essential oils found lemon and lemongrass oil could be used as sole carbon source for the bacteria; however cumin oil should only be used in small amounts as an inducer. They also suggested that these plant essential oils will result in a low cost and environmentally friendly approach for TCE bioremediation. Terpenes are present in the essential oil chemical composition of M. communis. These terpenes are a-pinene, camphene, b-pinene and limonene which comprise 34.45% of the total oil and after a year only 0.5% remained. In our study has found that terpenes disappearing easily from buried plant material and consumed by microorganisms or released in the soil. Myrtle terpenes could possibly participate in stimulation of organic pollutants biodegradation. Acknowledgements We would like to thank Dr. Ioanni Vogiatzaki, University of Reading, UK for suggestions on manuscript and linguistic improvements. References Adams, R., 2001. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy. Allured Publishing Co, Carol Stream, Illinois, pp. 63, 65, 70, 83, 84, 102, 145, 149, 161, 169, 171, 204, 212, 215, 216, 224, 236. Alhamd, L., Arakaki, S., Hagihara, A., 2004. Decomposition of leaf litter of four tree species in a subtropical evergreen broad-leaved forest, Okinawa Island, Japan. Forest Ecology and Management 202, 1e11. Ashnagar, A., Gharib, Naseri, N., Bayemani, A., 2009. Isolation and determination of the major chemical compounds present in essential oil of the leaves of myrtus plant grown in Khuzestan province of Iran. Asian Journal of Chemistry 21, 4969e4975. Berg, B., Hannus, K., Popoff, T., Theander, O., 1982. Changes in organic chemical components of needle litter during decomposition. Long-term decomposition in a Scots pine forest: I. (Pinus sylvestris). Canadian Journal of Botany 60, 1310e1319. Bisio, A., Ciarallo, G., Romussi, G., Fontana, N., Mascolo, N., Capasso, R., Biscardi, D., 1998. Chemical composition of essential oils from some Salvia species. Phytotheraphy Research 12, S117eS120.
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Davies, N.N., 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. Journal of Chromatography 503, 1e24. Focht, D.D., 1995. Strategies for the improvement of aerobic metabolism of polychlorinated biphenyls. Current Opinion in Biotechnology 6, 341e346. Gibbon, G.H., Pirt, S.J., 1971. The degradation of a-pinene by Pseudomonas PX1. FEBS Letters 18, 103e105. Gilbert, E.S., Crowley, D.E., 1997. Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. Strain B1B. Applied Environmental Microbiology 63, 1933e1938. Gunsalus, I.C., Marshall, V.P., 1974. Genetic regulation and mechanisms of P-450 oxygenase action. In: Survival and Toxic Environments. Academic Press, New York, San Francisco and London, pp. 295e313. Halligan, J.P., 1975. Toxic terpenes from Artemisia californica. Ecology 56, 999e1003. Harder, J., Foß, S., 1999. Anaerobic formation of the aromatic hydrocarbon p-cymene from monoterpenes by methanogenic enrichment cultures. Geomicrobiology Journal 16, 295e305. Hassiotis, C.N., 1997. Effects of plant secondary metabolites on soil bacteria and fungi populations. PhD thesis, University of Reading, UK. Hernandez, B.S., Koh, S.C., Chial, M., Focht, D.D., 1997. Terpene-utilizing isolates and their relevance to enhanced biotransformation of polychlorinated biphenyls in soil. Biodegradation 8, 153e158. Isidorov, V., Jdanova, M., 2002. Volatile organic compounds from leaves litter. Chemosphere 48, 975e979. Kanat, M., Alma, H.M., 2004. Insecticidal effects of essential oils from various plants against larvae of pine processionary moth (Thaumetopoea pityocampaSchiff) (Lepidoptera: Thaumetopoeidae). Pest Management Science 60, 173e177. Kaplan, D.L., Hartenstein, R., 1979. Problems with toluene and the determination of extracellular enzyme activity in soils. Soil Biology Biochemistry 11, 335e338. Kivanc, M., Akgul, A., 1986. Antibacterial activities of essential oils from Turkish spices and citrus. Flavour and Fragrance Journal 1, 175e179. Knobloch, K., Pauli, A., Iberl, B., Weigand, H., Weis, N., 1989. Antibacterial and antifungal properties of essential oil components. Journal of Essential Oil Research 1, 119e128. Margaris, N.S., Vokou, D., 1986. Microbial decomposition of smog organic pollutants. Bulletin of Environmental Contamination and Toxicology 36, 15e22. Marostica, M.R., Pastore, G.M., 2009. Limonene and its oxyfunctionalized compounds: biotransformation by microorganisms and their role as functional bioactive compounds. Food Science Biotechnology 18, 833e841. Massada, Y., 1976. Analysis of Essential Oil by Gas Chromatography and Spectrometry. John Wiley & Sons, New York, pp. 157e180. McLoughlin, E., Rhodes, H.A., Owen, M.S., Semple, T.K., 2009. Biogenic volatile organic compounds as a potential stimulator for organic contaminant degradation by soil microorganisms. Environmental Pollution 157, 86e94. Melillo, J.M., Aber, J.D., 1984. Nutrient immobilization in decaying litter: an example of carbon-nutrient interactions. In: Cooley, J.H., Golley, F.B. (Eds.), Trends in Ecological Research for the 1980s. Plenum Press, New York, pp. 193e215. Messaoud, C., Zaouali, Y., BenSalah, A., Khoudja, M.L., Boussaid, M., 2005. Myrtus communis in Tunisia: variability of the essential oil composition in natural populations. Flavour and Fragrance Journal 20, 577e582. Muller, W.H., Lorber, P., Haley, B., 1969. Volatile growth inhibitors produced by Salvia leucophylla: effects on oxygen uptake by mitochondrial suspensions. Bulletin Torrey Botanical Club 96, 89e96. Oh, E.T., Koh, S.C., Kim, E., Ahn, Y.H., So, J.S., 2003. Plant terpenes enhance survivability of polychlorinated biphenyl (PCB) degrading Pseudomonas pseudoalcaligenes KF707 labeled with gfp in microcosms contaminated with PCB. Journal of Microbiology and Biotechnology 13, 463e468. Panella, A.N., Dolan, C.M., Karchesy, J.J., Xiong, Y., Peralta-Cruz, J., Khasawneh, M., Montenieri, A.J., Maupin, O.G., 2005. Use of novel compounds for pest control: insecticidal and acaricidal activity of essential oil components from heartwood of Alaska yellow cedar. Journal of Medical Entomology 42, 352e358. Pausas, J.G., 1997. Litter fall and litter decomposition in Pinus sylvestris forests of the eastern Pyrenees. Journal of Vegetation Science 8, 643e650. Pereira, P.C., Cebola, M.J., Bernardo-Gil, M.G., 2009. Evolution of the yields and composition of essential oil from Portuguese myrtle (Myrtus communis L.) through the vegetative cycle. Molecules 14, 3094e3105. Pezhmanmehr, M., Dastan, D., Ebrahimi, S.N., Hadian, J., 2009. Essential oil constituents of leaves and fruits of Myrtus communis L. from Iran. Planta Medica 75, 1071e1079. Rajeswara Rao, B.R., Kaul, P.N., Bhattacharya, A.K., Rajput, D.K., Syamasundar, K.V., Ramesh, S., 2006. Comparative chemical composition of steam-distilled and water-soluble essential oils of South American marigold (Tagetes minuta L.). Journal of Essential Oil Research 18, 622e626. Rhodes, H.A., Owen, M.S., Semple, T.K., 2007. Biodegradation of 2,4-dichlorophenol in the presence of volatile organic compounds in soils under different vegetation types. FEMS Microbiology Letters 269, 323e330. Schwab, W., Davidovich-Rikanati, R., Lewinsohn, E., 2008. Biosynthesis of plantderived flavor compounds. The Plant Journal 54, 712e732. Shimoni, E., Baasov, T., Ravid, U., Shoham, Y., 2003. Biotransformations of propenylbenzenes by an Arthrobacter sp. and its t-anethole blocked mutants. Journal of Biotechnology 105, 61e70. Singh, G., Singh, O.P., Prasad, Y.R., de Lampasona, M.P., Catalan, C., 2002. Studies on essential oils, part 33: chemical and insecticidal investigations on leaf oil of Coleus amboinicus Lour. Flavor and Fragrance Journal 17, 440e442.
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Solyanikova, I.P., Travkin, V.M., Rybkina, D.O., Plotnikova, E.G., Golovleva, L.A., 2008. Variability of enzyme system of Nocardioform bacteria as a basis of their metabolic activity. Journal of Environmental Science Health B 43, 241e2522. Sombrero, C., 1992. Environmental control of oil production in Mediterranean plants. PhD thesis, University of Reading, UK. Steinbrecher, R., Hauff, K., Riissler, J., Diirr, M., Seufert, G., 1999. Monoterpene emission from soils in orange plantations of the Valencian Citrus Belt, Spain. Physics and Chemistry of the Earth (B) 24, 695e698. Stevenson, I.L., 1967. Utilization of aromatic hydrocarbons by Arthrobacter spp. Canadian Journal of Microbiology 13, 205e211. Suttinun, O., Muller, R., Luepromchai, E., 2009. Trichloroethylene cometabolic degradation by Rhodococcus sp. L4 induced with plant essential oils. Biodegradation 20, 281e291. Tandlich, R., Brezna, B., Dercova, K., 2001. The effect of terpenes on the biodegradation of polychlorinated biphenyls by Pseudomonas stutzeri. Chemosphere 44, 1547e1555.
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Decomposition process in the Mediterranean region. Chemical compounds and essential oil degradation from Myrtus communis Christos N. Hassiotis a, *, Diamanto M. Lazari b a b
Higher Technical University of Larissa, Department Natural Environment and Forestry, 43100 Karditsa, Greece Aristotle University of Thessaloniki, School of Pharmacy, Laboratory of Pharmacognosy, 54124, Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2010 Received in revised form 31 January 2010 Accepted 2 February 2010 Available online 24 April 2010
All aromatic plants emit volatile substances into the environment either during life cycle or during decomposition. These volatile constituents affect the top soil microflora, the nutrients recycle process and the vegetation establishment. Myrtus communis is a perennial aromatic shrub, rich in aromatic substances which can be found abundant in the Mediterranean. Fresh mature leaves of myrtle were used for this study using the litterbag technique. The essential oil content of the initial plant was 0.62% dry weight (dw) and after eighteen months burring dropped to 0.05%. The major oil compounds were 1,8-cineole (29.6%), a-pinene (24.7%) and myrtenyl acetate (10.6%). The essential oil degradation rates were similar under the relative small area of the investigation. Terpenes, esters and alcohols were released fast from buried material. The bacterial activity was induced by the presence of myrtle volatile oil. The only compounds that remained after eighteen months were 1,8-cineole and camphene. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Myrtus communis Essential oil Decomposition Bacteria Biorementation
1. Introduction Aromatic plants which contain essential oils are one of the major plant categories in the Mediterranean region. These plants have the capacity to synthesize, accumulate and emit volatiles that may act as aroma and flavor molecules due to interactions with living organisms. These low-molecular-weight substances derived from the fatty acid, amino acid and carbohydrate pools constitute a heterogenous group of molecules and cyclic structures bearing various functional groups (e.g. alcohols, aldehydes, ketones, esters and ethers) and also nitrogen and sulfur (Schwab et al., 2008). Almost all plants, and mainly the aromatic ones, emit volatile substances (Yang et al., 2009). The essential oil into aromatic plants represents 0.1e3% of the dry weight, and there are several ways that these secondary metabolites escape into the environment. Essential oils do not survive forever in plant material and the fade of the oil following leaf fall is a topic that requires more research. According to Margaris and Vokou (1986) the terpenoid emissions participate in photochemical reactions leading to aerosol production. Essential oil decomposition can occur even when the plant is placed in the dark. As Sombrero (1992) stated, the longer the plant remained in the dark the higher the drop in oil content, reaching approximately * Corresponding author. Tel.: þ30 6946 501110; fax: þ30 23920 92221. E-mail addresses: [email protected] (C.N. Hassiotis), [email protected] (D.M. Lazari). 0964-8305/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2010.02.007
40% in a period of twenty-four days. Plant essential oils are incorporated in plant material and follow the litter fall. Litter deposit depends primarily on the productivity of plant communities, which in turn are affected by climate, soil fertility, soil water retention and species composition (Pausas, 1997). The decomposition of the essential oil is achieved by the presence of microorganisms which need to have the enzymatic capacity to break down the organic compounds of the litter. The litter and its components constitute source of carbon and nutrients (Melillo and Aber, 1984). Plant essential oils constituents are insoluble or almost insoluble in water. The terpenoids which are characterized by their liability (Knobloch et al., 1989) have been found to interfere with enzymatic reactions of energy metabolism. The presence of essential oils or individual compounds derived from essential oils in the top soil layer has been found to enhance biodegradation (Tandlich et al., 2001; Isidorov and Jdanova, 2002; Rhodes et al., 2007; McLoughlin et al., 2009; Suttinun et al., 2009). The most prominent among the organisms known to attack hydrophobic residues are members of the genus Pseudomonas and Nocardia (Gunsalus and Marshall, 1974; Zorn et al., 2004; Solyanikova et al., 2008; Marostica and Pastore, 2009). It was also found (Stevenson, 1967) that Arthrobacter spp. are able to utilize a great number of aromatic hydrocarbons as their sole carbon source. There are a number of reports dealing with the ability of Arthrobacter in degrading aromatic structures (Shimoni et al., 2003). Gibbon and Pirt (1971) found that the volatile oil is decomposable by bacteria and that at least six species of Pseudomonas were able to degrade
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a-pinene, a common constituent of volatile oils. Kaplan and Hartenstein (1979) and Harder and Foß (1999) found that low concentrations of toluene in cultural media encouraged the growth of 19 species of bacteria and fungi which used toluene as their only source of carbon. On the other hand 1,8 cineole inhibits respiration (Muller et al., 1969) and alters the anatomy of the roots and seedling cells. As Halligan (1975) reported, camphor and 1,8-cineole were the two most toxic components and contribute to toxicity in the field. In the literature there are many reports for essential oil biosynthesis, essential oil composition and toxicity, biorementation using plant essential oils, for litter decomposition and the nutrient feedback (Halligan, 1975; Harder and Foß, 1999; Rhodes et al., 2007; Suttinun et al., 2009; Wannes et al., 2009). Despite on going research the role of essential oil degradation in plant litter in situ is ignored by many scientists. The rate of essential oil disappearing determines the remaining oil in plant litter especially when litter is accumulated year after year resulting in higher flammability, in microorganisms inhibition or activation, in nutrients immobilization, in allelopathic effects on other plants growth. The aim of this study was to investigate the degradation of essential oil during the decomposition process of myrtle (Myrtus communis), the way that the major compounds are released during that process and how bacteria populations are influenced by the presence of these compounds. 2. Materials and methods 2.1. Experimental area The area of the investigation was in the North part of Greece at Chalkidiki peninsula (latitude 40180 N; longitude 23 320 E). The peninsula and the experimental area is typically Mediterranean, characterized by hot dry summers and cool winters where the lower temperatures do not exceed five degrees below zero. The area of the investigation was rich in aromatic plant species. M. communis which was found in transitional vegetation communities was used for essential oil evaluation during decomposition process. 2.2. Experiment installation The litterbag technique (Alhamd et al., 2004) was used to evaluate the litter decay, the fade of the essential oil content and its chemical compounds disappearing. Freshly abscised leaves of M. communis were collected in the study area (8000 m2) by shaking 20 Myrtle shrubs over a large net at the end of July where the essential oil content is quite high. Any debris was carefully removed from each collection. The raw material was mixed, the essential oil was extracted and measured and the oil composition was evaluated by GCeMS analysis. Four kilos of raw material were used and the samples were put in litterbags. Leaf samples (about 20 g fw) were enclosed in terylene net bags (16 10 cm). The mesh size (1 mm2) was small enough to prevent the loss of leaf fragments, but large enough to allow aerobic microbial activity and the entry of small soil animals. Twelve replicates, of sixteen bags, in total 192 litterbags, were installed in the experimental area under twelve myrtle shrubs which were selected randomly, on the 1st of August 2006. The experiment was carried out for 18 months which including one month of preparations. 2.3. Sample collection The first sampling was on the 1st of September 2006 (twelve litterbags, one of each replication) and then periodically, every month. The extraction of the oil was achieved by organic solvent (Fig. 1). In the solvent extraction the plant material was put into a plastic pipette tip and was kept by nonabsorbent cotton from both sides. The cotton on the bottom filters the solvent and regulates the flow through the plant
Fig. 1. Schematics for essential oil solvent extraction. The plant material is trapped by nonabsorbent cotton. The macerate is washed with solvent (diethyl ether) in order to extract the oil.
material thus controlling the time of extraction. The cotton on the top prevents accidental losses of the plant material when applying the solvent. The extraction medium was diethyl ether with 104 M (M) of n-tetradecane to act as an internal standard for any losses (Hassiotis, 1997). Approximately 2 g of myrtle was put into the capsule after a precise weighting and covered by the top cotton. 5 ml of solvent (diethyl ether) was added on the top and left to flow through the plant sample, percolating the essential oil. The washing lasts about 15 min and three successive washings were taken. At the end of each washing an elastic tube was fitted on the open top, which was connected with an air pump. The air pressure breaks the structures which are resistant to the solvent treatment. The dilution was transferred and left at room temperature for evaporation (in a fume cupboard) and the essential oil was measured. Three samples of each litterbag were solvent extracted for precise essential oil evaluation. Moreover, 2 g from the litterbags were used to calculate the humidity of the samples and to express the oil content per dry weight. 2.4. Essential oil analysis The composition of the volatile constituents was established by GCeMS analysis. GCeMS analysis were performed on a Shimadzu GC-2010 e GCMS-QP2010 system operating in EI mode (70 eV) equipped with a split/splitless injector (230 C), a split ratio 1/30, using a fused silica HP-5MS capillary column (30 m 0.25 mm (i.d.), film thickness: 0.25 mm). The temperature program was from 50 C (5 min) to 290 C at a rate of 4 C min1. Helium was used as a carrier gas at a flow rate of 1.0 mL min1. Injection volume of each sample was 1 mL. Retention indices for all compounds were determined according to the Van den Dool approach (Van den Dool and Kratz, 1963), using n-alkanes as standards. The identification of the components was based on comparison of their mass spectra with those of NIST21 and NIST107 (Massada, 1976) and those described by Adams (2001) as well as by comparison of their retention indices with literature data (Bisio et al., 1998; Davies, 1990). All data have been tested for statistically important differences with analysis of variance test using SPSS 16.0 for windows. 2.5. Bacteria analysis Three samples of 1 g per litterbag were collected, cut into small pieces (approximately 5 mm) and dissolved into 100 ml Ringers
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solution (500 ml distilled water plus one tablet for stock Ringers solution). After 5 min in suspension, one ml of each solution was transferred into a further 100 ml Ringers solution and a serial dilution of one more step carried out. After the dilution the total concentration was equivalent to 1g 106 of myrtle litter per ml Ringers solution. Agar substrate was prepared a day before using agar powder, bacteriological peptone, and yeast extract in the ratio 1.5%, 1%, 1% respectively. Distilled water was used for the dilution. In order to avoid any contamination the agar was sterilized in an autoclave at 120 C and 2 atm pressure for 30 min. The petri-dishes (8 cm in diameter) were filled with this agar substrate which was left for 24 h period to check for contamination. After checking, the clear petri-dishes were used for bacteria cultivation. One ml was pipetted from the final Ringers dilution (106) and was spread on the agar surface (three petri dishes were used for each sample). A sterilized glass spreader was used for the drop spreading and then rotated clockwise and anticlockwise to get homogenous distribution of the suspension to the medium. The plates were marked and incubated in a sterilized chamber, at 26 C. The number of bacterial colonies was counted 48 h, after spreading and recorded following the colony forming unit (CFU/g) standards. The bacterial colonies were counted from nine petri dishes of each litterbag, analyzed by using StatistiXL Version 1.8 add-in for MS Excel (Nedlands, Western Australia) and presented as mean standard deviation. 3. Results and discussion 3.1. Bacterial colonies The mean number of bacterial colonies developed on agar medium 48h after spreading is presented in Fig. 2. At the date of experiment installation on the 1st of August the average bacterial colonies in myrtle leaf material was 198.1 106 g1. After one month the bacterial colonies in burring myrtle increased 27.7% and after two months reached up to 288.2 106 g1 in October and 286.4 106 g1 in November. This period apart from fresh myrtle “food” for bacteria coincides with favorable weather conditions. The bacterial activity started to decline from December to August, where the minimum activity was recorded. On the 1st of August 2007, the bacteria numbers dropped at 103.1 106 g1 and then slightly increased up to
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Fig. 2. Essential oil percentage derived from buried M. communis (left y axis). The hi-low lines present oil fluctuation between replications. Right y axis: Bacterial colonies (standard deviation) developed from litter of M. communis. The plant material was buried from August 2006 to December 2007.
the end of the study, reaching at 122.4 106 g1. Comparing the period of October and November 2006 with the respective period of October and November 2007 (with similar weather conditions) it is revealed 138% higher bacterial activity in the first period. This observation leads us to the conclusion that fresh buried myrtle material (two months old) was used as carbon source and enhanced bacterial activity. 3.2. Essential oil decomposition The essential oil content obtained in our study is presented in Fig. 2. During the period of the investigation the essential oil content of buried Myrtus communis presented a remarkable decrease. After a period of eighteen months the essential oil content was reduced at about 93% (from 0.62% to 0.05%). This means that almost the total amount of myrtle oil has been disappeared during the period of eighteen months. Even after this great oil losses, a small amount of oil remained into plant material at the end of the study. Analysis of variance which was performed in order to test the remaining essential oil into plant material during the study resulted in significant differences between the dates of samplings (P < 0.01). In other words, in every new monthly sampling there were remarkable losses of essential oil from buried myrtle. The differences in essential oil content between experimental sites, for the same dates of samplings, were also tested by ANOVA and they did not present any differences leading to the conclusion that in a small area, as the one of this study, the essential oil degradation rates were presented as more or less uniform. The aim of the twelve replications of litterbags was targeted to ensure precise results in oil disappearance and not to investigate the influence of locality in decomposition rates. While the experiment installation was in August the initial oil losses recorded in October and up to December. From January to March there is a slow down whereas from April to June there is an increase in the rhythm that the oil disappeared. The same pattern observed from July to September and then increased again up to the end of the year (Fig. 2). There were differences in the two periods of the study, October to December 2006 and January to March 2007. In the first period a remarkable oil disappearance is shown, which is possibly correlated with the start of rainfalls in the autumn and higher bacterial activity, whereas in the second group, even with higher precipitations, the oil losses are rather low. This observation supports the opinion that the most important, among environmental parameters affecting decomposition in Mediterranean, is the temperature during summer and winter. Even though the higher precipitations in January, February and March the oil losses are restricted due to the low temperatures which in turn affect the bacterial and microbial activity. Berg et al. (1982) reported that the constituents of the litter can be divided into three broad groups which begin their net mass loss at different stages of decomposition. Concerning the essential oil degradation or disappearance, Knobloch et al. (1989) and later Rajeswara Rao et al. (2006) reported that the slight water solubility of terpenes would allow some leaching loss of these components and moreover the biological effect of the oil constituents is related to their solubility in water and/or their solubility in membrane lipids. The solubility of terpenes, in addition to favorable climatic conditions for microbial activity is possibly the reason why a remarkable oil loss is presented from October to December. The present results do not indicate that losses are due to evaporation. At the date of experiment installation and for a month period (August to September 2006) where the oil content was higher in myrtle and the temperatures very high (day average 26.7 C) did not record oil disappearance. It is indicated from this study that the essential oil disappearance is depending from bacterial activity which in turn is limited by environmental factors such as temperature and rainfalls.
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The microorganisms during summer are limited by high temperatures and during winter by low. The average temperature of the coldest month is 5.6 C, whereas that of the hottest month is 26.7 C. August is the driest month of the year with an average rainfall of 14.3 mm (data of the meteorological station of Aristotle University of Thessaloniki, the nearest to the study area). Oil leaching and possibly immediate consumption by bacteria is recorded only during the first wet period after installation whilst the disappearance through evaporation is almost zero. The knowledge of essential oil amount into buried plant material has one more ecological aspect because essential oils have also major roles in presence of other organisms apart from microorganisms. There are several reports on the effect of essential oils and its chemical compounds on other organisms. Singh et al. (2002) working with oil from Coleus amboinicus found it to be insecticidal to white termites (Odontotermes obesus Rhamb.) with 100% mortality at a dose of 2.5 102 mg cm3 for 5 h exposure. Actually the oil from C. amboinicus was also more active than the synthetic insecticides, Thiodan and Primoban-20, against termites. Kanat and Alma (2004) indicated that the essential oils from nine plant species and sulfate turpentine were effective against the larvae of Thaumetopoea pityocampa. Panella et al. (2005) reported the ability of essential oil components to kill arthropods at relatively low concentrations represents an alternative to the use of synthetic pesticides for control of disease vectors. 3.3. Essential oil analysis The essential oil analysis demonstrated that forty seven compounds comprise 98.8% of the total oil. Seventeen compounds presented percentage more than 0.1% w/w (Table 1) comprising the 94.80% of the total oil. The two major compounds were 1,8-cineole and a-pinene with an average percentage of 29.60% and 24.71% respectively. Other predominant compounds were myrtenyl acetate (10.60%), limonene (6.91%), a-terpineol (6.10%) and linalool (4.30%). The results of essential oil analysis of this study for myrtle in Greece are in agreement with relative work done recently in the Mediterranean region (Messaoud et al., 2005; Wannes et al., 2007; Pereira et al., 2009 and Wannes et al., 2009) as well as around the world (Ashnagar et al., 2009; Pezhmanmehr et al., 2009). Four groups of compounds participate in the chemical composition of Table 1 Chemical composition of Myrtus communis essential oil used for the experiment. Compoundsa
RIb
Composition (%) SDc
a-Pinene
932 946 974 1028 1029 1100 1190 1197 1228 1256 1257 1324 1351 1355 1366 1385 1402
24.71 0.81 2.02 6.91 29.60 4.30 6.10 0.99 1.16 0.80 1.49 10.60 0.99 0.87 1.13 0.40 1.92 94.80
Camphene b-Pinene Limonene 1,8-Cineole Linalool a-Terpineol Myrtenol Nerol Geraniol Linalyl acetate Myrtenyl acetate a-Terpinyl acetate Eugenol Neryl acetate Geranyl acetate Methyl eugenol TOTAL
0.76 0.02 0.04 0.34 1.19 0.25 0.33 0.04 0.08 0.02 0.04 0.41 0.04 0.03 0.03 0.04 0.06
a Only compounds contributing more than 0.1% of the total oil are presented. Compounds listed in order of elution from an HP-5MS column. b Retention indices as determined on an HP-5MS column using a homologous series of n-alkanes. c Standard deviation.
359
myrtle oil. These are terpenes (34.4%), esters (14.6%), alcohols (16.2%) and ethers (29.6%). The way that the individual chemical compounds are released from buried M. communis essential oil are presented (compound average) in Table 2. All compounds during this study of eighteen months showed different decrease rates. There are remarkable losses for each compound percentage for all experimental sites. The only compounds remaining into plant material after a year period were camphene, 1,8-cineole and methyl eugenol while at the end of the study only camphene (0.1%) and 1,8-cineole (4.8%) were present in the remaining oil. We could infer that the remaining 1,8-cineole (4.8%) is due to the high initial percentage (29.6%) of this compound in the total oil although this did not also happen in a-pinene. The initial 24.7% of this compound in the total oil disappeared within a year period. This is an indication that the initial high percentage of a compound in an essential oil cannot be a reliable index for the presence of this compound in plant litter after a half or a year period and generalizations cannot be done. In a previous study, Hassiotis (1997) working with Laurus nobilis essential oil degradation and compounds release found that after fifteen months period the only compounds remained in the final essential oil were 1,8 cineole and camphor. Most of the compounds disappeared easily from the plant litter mainly by microorganism consumption and less by leaching or evaporation. The importance of microorganisms to decompose natural aromatic structures has been reported by Harder and Foß (1999). In 1988, Vokou and Margaris found that under favorable climatic conditions (sufficient moisture) soil microorganisms have the capacity to decompose natural products, such as secondary metabolites, at a rate of at least 1.7 g m2 d1. Even 1,8-cineole, which is reported as very toxic to microorganisms (Kivanc and Akgul, 1986) can be degraded by microorganisms. This cineole toxicity possibly resulted to be present in the final plant litter after eighteen months of degradation. The way that the groups of compounds are released during decomposition is presented in Fig. 3 with intervals of four months. There is a remarkable decrease in groups of terpenes, alcohols and esters whereas ethers (mainly 1,8 cineole) degradation rates are slow. The total amount of terpenes, alcohols and esters in the initial material was 65.2%, in December 2006, 28.9%, in April 2007, 7.0% and in August 2007 only 0.1%. The fast degradation of these groups of compounds is positively correlated with the high bacterial activity in the first eight months of the experiment. The correlation between fast (terpenes, alcohols and esters) and slow (ethers) decomposable groups of compounds in myrtle oil and bacterial colonies developed from myrtle litter are shown in Fig. 4. It is observed a two way relation between rapid released groups of compounds and bacterial activity. The fast release of the compounds is due to bacterial activity or the higher bacterial activity is induced by the presence of easily decomposable compounds. The high bacterial activity seems not to influence the degradation of ethers (mainly 1,8-cineole) whose rate is slow. 1,8-cineole is known as compound causes bacteria inhibition. This inhibition did not manifest up to January 2007 where equilibrium between faster and slower degraded compounds were observed. From March to August 2007, where the easily decomposable compounds have almost disappeared, the pattern of bacteria follows the pattern of ethers. It is worth noting that for that period as the temperature increased rainfall decreased. The rapid release of terpenes has an ecological approach since they induce degrading bacteria. There is speculation that volatile organic compounds (VOCs) emitted within soil either by roots or by decaying biomass may enhance the biodegradation of persistent organic pollutants through cometabolism. Several studies have demonstrated the role of monoterpene compounds in the induction or enhancement of polychlorinated biphenyl (PCB) biodegradation. These many plant-derived chemicals, including also those
C.N. Hassiotis, D.M. Lazari / International Biodeterioration & Biodegradation 64 (2010) 356e362 40
groups of compounds (%)
1.1 0.9 0.7 0.5 0.3 0.2 0.1 0.1 e e e e e e e e e
0.4 0.2 0.1 0.1 0.1 e e e e e e e e e e e e
1.9 1.6 1.5 1.5 1.5 1.4 1.3 1.1 0.9 0.8 0.8 0.6 0.4 0.3 0.3 e e
35 30 25
ethers terpenes
20
alcohols esters
15 10
0.9 0.9 0.9 0.7 0.6 0.5 0.4 0.3 0.2 0.1 e e e e e e e
eugenol
neryl acetate
geranyl acetate
methyl eugenol
360
0
Apr-07
Aug-07
Dec-07
Fig. 3. Groups of chemical compounds participating in chemical composition of Myrtus communis essential oil. The plant material was buried for seventeen months. The highest and the lower amount of each group are presented by Y error bars.
generated from root turnover, stimulate microorganisms to biodegrade xenobiotics (Isidorov and Jdanova, 2002). Steinbrecher et al. (1999) attempted to evaluate the terpenoid emissions working with Citrus species and found that were in the range of 1500e3000 pmol mm2 s1 from soils under the trees covered with litter, and of 10e100 pmol mm2 s1 from bare soils between rows. The origin of emissions from leaf and fruit litter was experimentally confirmed by removal of the uppermost soil layers. Focht (1995) proposed that plant terpenes might be the natural substrate for PCB oxidation, rather than biphenyl. Later Hernandez et al. (1997) demonstrated that soils enriched with orange peel, ivy leaves, pine needles or eucalyptus leaves resulted in 105 times more biphenyl utilizers (108 g1) than unamended soils (103 g1) simultaneously inducing Aroclor 1242 degradation. Gilbert and Crowley (1997) screened several terpenoid compounds for their ability to induce PCB biodegradation in Arthrobacter sp. strain B1B and resulted that s-carvone, the principal chemical component of
300
6
groups of compounds (%)
200 40 150 30 100 20
50
0 Dec-07
Oct-07
Nov-07
Sep-07
Jul-07
Aug-07
Jun-07
Apr-07
May-07
Mar-07
Jan-07
Feb-07
Oct-06
Sep-06
Dec-06
10
Aug-06
24.7 22.4 19.4 14.9 10.7 7.5 5.2 3.4 2.1 1.1 0.4 0.2 0.1 e e e e
250
50
Nov-06
0.8 0.7 0.7 0.6 0.6 0.6 0.6 0.5 0.5 0.4 0.3 0.3 0.3 0.2 0.2 0.1 0.1
2.0 1.8 1.6 1.2 0.9 0.6 0.4 0.3 0.2 0.1 e e e e e e e
6.9 7.0 6.7 5.7 4.5 3.5 2.7 2.0 1.4 0.8 0.3 0.1 0.1 e e e e
29.6 29.8 28.6 25.3 23.5 23.1 23.0 21.6 20.5 16.4 13.4 12.2 11.7 8.7 6.7 5.1 4.8
60
0
Aug-06 Sep-06 Oct-06 Nov-06 Dec-06 Jan-07 Feb-07 Mar-07 Apr-07 May-07 Jun-07 Jul-07 Aug-07 Sep-07 Oct-07 Nov-07 Dec-07
1,8-cineole limonene
b-pinene camphene
a-pinene
Compounds of Myrtus communis essential oil (%)
Terpenes & Alcohols & Esters Ethers Bacterial colonies
-1
6.1 5.8 5.3 4.3 3.3 2.4 1.8 1.2 0.8 0.4 0.2 0.1 e e e e e 4.3 3.9 3.4 2.6 1.9 1.3 0.9 0.6 0.4 0.2 0.1 e e e e e e
Dec-06
bacterial colonies x 10 g dry weight
a-terpineol linalool
1.0 1.0 0.8 0.6 0.5 0.4 0.3 0.2 0.1 e e e e e e e e
1.2 1.1 0.9 0.6 0.5 0.4 0.2 0.2 0.1 e e e e e e e e
0.8 0.8 0.7 0.5 0.4 0.3 0.2 0.2 0.1 e e e e e e e e
1.5 1.0 0.7 0.4 0.2 0.1 0.1 e e e e e e e e e e
10.6 8.1 5.8 4.1 2.8 1.9 1.2 0.6 0.2 0.1 e e e e e e e
1.0 0.7 0.5 0.3 0.2 0.1 0.1 e e e e e e e e e e
Aug-06
70
Date
Table 2 Average percentage of the main compounds found in essential oil of buried Myrtus communis.
myrtenol
nerol
geraniol
linalyl acetate
myrtenyl acetate
a-terpinyl acetate
5
Fig. 4. Release rates of terpenes, alcohols and esters in comparison with release rate of ethers derived from M. communis essential oil (left y axis). Right y axis: Bacterial colonies (standard deviation) developed from litter of M. communis. The plant material was buried from August 2006 to December 2007.
C.N. Hassiotis, D.M. Lazari / International Biodeterioration & Biodegradation 64 (2010) 356e362
spearmint (Mentha spicata), induced the biotransformation of 62% of Aroclor 1242, or 26 of 32 identified peaks. Tandlich et al. (2001) used carvone and limonene to stimulate biodegradation of Delor 103 (another commercial mixture of PCBs) by Pseudomonas stutzeri. Additional PCB congener removal occurred after supplementing with terpenes compared with control-grown cultures. In 2003, Oh et al. examined the ability of terpenes to prolong the survival of a known PCB-degrading bacterium, Pseudomonas pseudoalcaligenes KF707, in soil. The addition of 50 mg l1 p-cymene or 50 mg l1 a-terpinene increased KF707 survival by 10e100 fold compared with biphenyl supplemented and control mesocosms. Rhodes et al. (2007) concluded that the degradation of 2,4-dichlorophenol by indigenous microorganisms is greater in those soils amended and ‘aged’ with monoterpenes, (a-pinene, limonene and p-cymene) than in freshly spiked or control soils. She also suggested that the remediation of contaminated soils may be promoted by stimulating indigenous microorganisms either through planting monoterpeneemitting vegetation, particularly pine species, or through direct application of natural substances with high monoterpene content, for example pine needle litter or citrus waste. Lately McLoughlin et al. (2009) suggested that monoterpenes can stimulate the biodegradation of 2,4-DCP by indigenous soil microorganisms and that monoterpene amendment in soils is an effective strategy for removing organic contaminants. Therefore, it is feasible to suggest that there is potential for in situ remediation of contaminated soils through the stimulation of indigenous microorganisms through applications of exogenous isoprenoid such as terpene rich plant residues or the planting of isoprenoid-emitting vegetation. Recently Suttinun et al. (2009) working with induction of TCE (Trichloroethylene)-degrading enzymes with essential oils found lemon and lemongrass oil could be used as sole carbon source for the bacteria; however cumin oil should only be used in small amounts as an inducer. They also suggested that these plant essential oils will result in a low cost and environmentally friendly approach for TCE bioremediation. Terpenes are present in the essential oil chemical composition of M. communis. These terpenes are a-pinene, camphene, b-pinene and limonene which comprise 34.45% of the total oil and after a year only 0.5% remained. In our study has found that terpenes disappearing easily from buried plant material and consumed by microorganisms or released in the soil. Myrtle terpenes could possibly participate in stimulation of organic pollutants biodegradation. Acknowledgements We would like to thank Dr. Ioanni Vogiatzaki, University of Reading, UK for suggestions on manuscript and linguistic improvements. References Adams, R., 2001. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy. Allured Publishing Co, Carol Stream, Illinois, pp. 63, 65, 70, 83, 84, 102, 145, 149, 161, 169, 171, 204, 212, 215, 216, 224, 236. Alhamd, L., Arakaki, S., Hagihara, A., 2004. Decomposition of leaf litter of four tree species in a subtropical evergreen broad-leaved forest, Okinawa Island, Japan. Forest Ecology and Management 202, 1e11. Ashnagar, A., Gharib, Naseri, N., Bayemani, A., 2009. Isolation and determination of the major chemical compounds present in essential oil of the leaves of myrtus plant grown in Khuzestan province of Iran. Asian Journal of Chemistry 21, 4969e4975. Berg, B., Hannus, K., Popoff, T., Theander, O., 1982. Changes in organic chemical components of needle litter during decomposition. Long-term decomposition in a Scots pine forest: I. (Pinus sylvestris). Canadian Journal of Botany 60, 1310e1319. Bisio, A., Ciarallo, G., Romussi, G., Fontana, N., Mascolo, N., Capasso, R., Biscardi, D., 1998. Chemical composition of essential oils from some Salvia species. Phytotheraphy Research 12, S117eS120.
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