Biodegradation of heavy crude oil Maya using spent compost and sugar cane bagasse wastes

Chemosphere 68 (2007) 848–855 www.elsevier.com/locate/chemosphere

Biodegradation of heavy crude oil Maya using spent compost and sugar cane bagasse wastes M.R. Trejo-Herna´ndez a

a,*

, A. Ortiz a, A.I. Okoh b, D. Morales a, R. Quintero

a

Centro de Investigacio´n en Biotecnologı´a, Universidad Auto´noma del Estado de Morelos, Av. Universidad 1001, Cuernavaca C.P. 62209, Morelos, Mexico b Department of Biochemistry and Microbiology, University of Fort Hare, Alice, South Africa Received 20 November 2006; received in revised form 9 February 2007; accepted 9 February 2007 Available online 28 March 2007

Abstract Experiments were carried out to evaluate the use of some agroindustrial wastes as supports in solid state cultures for the biodegradation of crude oil Maya in static column reactors over 15–20 days periods. Spent compost and cane bagasse wastes showed superior qualities over peat moss waste as support candidates with the advantage that they contain appreciable densities of autochthonous microorganisms in the order of 102 cfu g 1. Mercuric chloride (2%) was able to completely inhibit growth of these microfloras. Biodegradation was enhanced in the presence of the IMP consortium and highest when microflora from cane bagasse only was the bioaugmentation partner (180.7 mg kg 1 day 1). Combination of these waste materials (3:1 ratio, respectively) was observed to significantly biodegrade the crude oil by approximately 40% in 15 days from an initial concentration of 10 000 mg kg 1 with a four order of magnitude increase in microbial density during this period. Spent compost and cane bagasse wastes are veritable solid support candidates for use in the biodegradation of crude oil polluted systems.  2007 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Crude oil; Agroindustrial wastes

1. Introduction In the last few decades of the 20th century, the pollution of the ecosystem has become a matter of increasing international concern. Major contributors to environmental pollution include the chemical industries, run-offs from agriculture activities, as well as effluents from urban areas to mention a few, as a consequence of extensive industrialization which has also caused a deepened over reliance on petroleum hydrocarbon as source of energy (Faber and Krieg, 2002). This has resulted in extensive exploration of new sources of crude oil to meet the world’s ever increasing demand. The attendant negative consequences of these explorative and exploitative activities have been the pollution of the environment especially through spillages and

*

Corresponding author. Tel.: +52 777 329 7057; fax: +52 777 329 7030. E-mail address: [email protected] (M.R. Trejo-Herna´ndez).

0045-6535/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.02.023

accidents (Thouand et al., 1999; Okoh et al., 2002; Okoh, 2006). A wide range of practices are employed for management of crude oil pollution. The technological diversity is the result of widely varying geological, climatological, ecological, topographic, economic, geographic, and age differences among drilling and production sites. Current practices of oil wastes management include the use of reserve pits for drilling wastes; land spreading of reserve pit contents; and disposal of aqueous effluents through Class II underground injection wells (EPA, 1988, 2002). Biological alternatives are also applied for treating pollution of contaminated areas, although, this and the other management practices are features common only in developed countries. Several studies have reported on the potentials of composted materials in the biodegradation of chemical pollutants. For example, Wischmann and Steinhart (1997) reported that compost materials enhanced the degradation of a synthetic PAH/N-PAH mixture enriched in recalcitrant compounds

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with four and five fused rings – particularly benzo[a]pyrene. Also, Meyer and Steinhart (2000) used the autochthonous microflora of the soil/compost mixture to assess the effects of hetero-PAHs on the biodegradation of typical tar oil PAHs in a soil material from an AhA1-horizon supplemented with compost under aerobic conditions. In our previous study in Mexico (Trejo-Hernandez et al., 2001) we demonstrate that the residual compost in Agaricus bisporus farming,is a potential source of laccase that could become a cost-effective waste management alternative for some phenolic compounds. That report was an impetus in our on going effort at establishing a cost effective waste management strategy for the bioremediation of crude oil contaminated system in Mexico, of which this current study was a part. In the biological treatment of hazardous wastes, the use of organic support materials has been recommended (Potter et al., 1999). This strategy, which has been used to reduce large-scale contamination problems, involves the addition of lignocellulosic materials that increase water retention and porosity of contaminated soils. Within this context, different materials (peat moss, volcanic rock, compost, straw, bagasse, etc.) have been used which help to accelerate degradation of toxic compounds (Rao et al., 1995). However, this process suffers from the limitation that the lignocellulosic material is also consumed during the degradation process, and therefore the initial texture and porosity of the material is lost. Given this limitation, it is necessary to modify this process to prevent degradation of the organic material and the consequent loss of solid matrix texture. In studies of solid state culture it is common to use lignocellulosic materials, since they favor microbial growth on surface area and within the solid matrix as a result of their porosity, thus increasing water retention, and favoring gas transfer (Lonsane et al., 1985; Stegmann et al., 1991). The use of organic materials in hydrocarbon biodegradation in contaminated soils offers advantages to the process, since they allow in the first place, reduction of mass transfer problems while increasing porosity and moisture retention of solid matrix (Trejo-Hernandez et al., 1993; Mahro et al., 1994). Also, these materials are a natural support for native microflora such as the fungus A. bisporus that can degrade lignin or lignocellulose-type materials. The potential use of edible fungi from wood white rot and ectomycorrhizas, has already been demonstrated in biodegradation of different contaminants such as phenol, chlorophenol, anilines, pesticides, nitrotoluene and hydrocarbons (Breitung et al., 1996; Plestch et al., 1999; TrejoHernandez et al., 2001). Different authors demonstrated that the addition of the mature compost in the bioremediation processes accelerate the biodegradation and stabilization of toxic compounds (Benoit and Barriuso, 1995; Cole et al., 1995; Hupe et al., 1996). In this paper, we report the biodegradation of the heavy crude oil Maya on solid state cultures using spent compost and sugar cane bagasse wastes as solid support.

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2. Materials and methods 2.1. Reagents Chromatographic-grade dichloromethane and anhydrous sodium sulfate were purchased from J.T. Baker S.A. 2.2. Substrates The heavy crude oil Maya was kindly provided by the Mexican Petroleum Institute (IMP). This oil is composed of 30% asphalthenes, 26.9% aliphatic hydrocarbons, 35.5% aromatic hydrocarbons and 3.6% polar hydrocarbons as estimated according to the description of Dutta and Harayama (2001) with modification. 2.3. Agroindustrial solid wastes The agroindustrial solid waste materials used were selected based on their abundance and availability. Spent compost wastes were obtained from a plant that produces edible mushrooms (Marvel S.A. de C.V.) in Texcoco, Mexico. This material is a waste from the production process of edible mushrooms and is composed mainly of wheat straw. Sugar cane bagasse wastes were obtained from Zacatepec Sugar Mill, in the State of Morelos, Mexico. Both materials were ground and screened through a sieve of mesh size of 2 mm. The nitrogen, phosphorous and organic matter contents of the waste materials were determined as previously described (Ortiz et al., 1993). Peat moss was purchased from Canadian Sphagnum Peat Moss Association. 2.4. Microbial consortium The microbial consortium used in the bioaugmentation experiments was isolated by the Mexican Petroleum Institute (IMP), from soils contaminated with oil wastes from crude oil exploration facilities of PEMEX (Mexican Oils) in Minatitla´n, Veracruz, Mexico. Six bacteria species belonging to Pseudomonas, Bacillus, Klebsiella and Serratia genera constitute the microbial consortium. This consortium was grown in a modified mineral medium of Ka¨stner et al. (1994) with crude oil (1%) as carbon source. The inoculum was grown at pH 7.0 with orbital agitation at 175 rpm for a period of 8 days at 28 C. 2.5. Degradation experiments The spent compost or cane bagasse wastes was artificially contaminated with the crude oil (10 000 mg kg 1) dissolved in 1 ml of dichloromethane and the mineral medium was added. Biodegradation studies were carried out in labscale tubular reactors with 125 g of moistened material (60–65% moisture), pH 7, aeration 10 ml min 1 during an incubation period of 15 days at 28 C. Spent compost and cane bagasse wastes ratio was 3:1 with a C:N:P ratio

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of 100:11:5. In the bioaugmentation experiments the inoculum used was IMP consortium (104 cfu g 1 dw) and the experimental setup were as follows: (i) Inhibited control (addition of HgCl2 2% dw); (ii) IMP consortium inoculated on sterilized solid materials wastes; (iii) IMP consortium inoculated on solid materials wastes with natural microflora; (iv) natural microflora from solid waste materials; (v) IMP consortium inoculated on solid wastes materials with only natural microflora from compost wastes; and (vi) IMP consortium inoculated on solid wastes materials with only natural microflora from sugar cane bagasse wastes. The tubular reactors used were made of glass (30 cm in height · 4.6 cm inner diameter), and equipped with a moisturized air supply device. This device consists of a container with sterilized water, connected to a dry air supplier. For the inhibition of natural microflora of the solid wastes materials, we subjected the materials to different physicochemical treatments such as sodium azide (1%) addition; perchloric acid (7%) addition; UV light exposure (5 h); addition of mercuric chloride (2%); and heat sterilization to select the most efficient for use in abiotic control. To assess the contribution of indigenous microflora to the biodegradation process, the appropriate set of solid waste materials were sterilized by three runs of autoclaving at 100 C for 25 min. All experiments were carried out in triplicate. At the end of the evaluation mercuric chloride (2%) was selected for the abiotic control experiment. 2.6. Kinetic studies Kinetic studies were carried out in column reactors regimes of three reactors per incubation period, and sampling was done every five days over a 20 days reaction period. Experimental conditions include: 125 g of material (spent compost or cane bagasse wastes) artificially contaminated with 10 000 mg kg 1 of crude oil Maya; pH 7.0; aeration at 30 ml min 1; and initial viable cell count 103–104 cfu g 1 dw. Abiotic controls were also set up in parallel, and all experiments were done in triplicate. Samples were analyzed for residual hydrocarbon and viable cell count. 2.7. Analytical techniques 2.7.1. Crude oil extraction and quantification Solid samples were extracted using the modified EPA 418.1 technique (EPA, 1991). Two grams wet weight of solid material were mixed with 1 g anhydrous sodium sulfate and 0.5 g silica gel, and extracted with chromatographic-grade dichloromethane using vacuum filtration. Sample extracts (1 ll) were injected and analyzed in a gas chromatograph Hewlett Packard Mod. HP-5890 equipped with a flame ionization detector (FID), and a rubber capillary column of HP-Methyl-silicon, (25.5 m long · 0.32 mm inner diameter). The injection was carried out with split 1:10. Helium was used as gas carrier with a flow of

29 ml/min. The injector was kept at a temperature of 270 C. Initial temperature was 90 C with an initial gradient of 2 C min 1 up to 150 C and a second gradient from 150 to 280 C/10 C min 1. The detector’s temperature was 300 C. 2.7.2. Viable count The viable cell count of total heterotrophs was determined by standard spread plate technique (Seeley and VanDenmark, 1981). One gram each of the samples was suspended in sterile physiological saline and diluted serially using sterile peptone water, and 200 ll of the diluted samples was then plated onto the surface of plate count agar and incubated aerobically at 37 C for 48 h. At the end of incubation, plates containing between 30 and 300 colonies were selected for counting and expressed as colony forming units per gram dry weight (cfu g 1 dw). 2.7.3. pH determination Determination of pH was carried out by adding 2 g of solid sample and 10 ml of sterilized water in a 50 ml Erlenmeyer flask. The suspension was stirred at 100 rpm for 30 min on an orbital agitator (Trejo-Hernandez et al., 1993). pH was measured using an Orion potentiometer. 2.7.4. Elemental and moisture content analysis Moisture content of all the samples was determined by the method described by Saucedo-Castan˜eda et al. (1994). Determination of N, P and organic matter were done as previously described (Ortiz et al., 1993). 2.7.5. Residual hydrocarbon estimation Total residual hydrocarbon was estimated by a gravimetric method as in accordance with our previous description (Okoh et al., 2001) using dichloromethane as extraction solvent. Approximately 95% oil recuperation was achieved by this method. 3. Results 3.1. Evaluation of solid support candidates The solid wastes materials were evaluated based on their moisture retention ability and hydrocarbon recovery efficiency using dichloromethane as extraction solvent. Additionally, nitrogen, phosphorous and organic matter contents of the materials were determined. Results obtained (Table 1) from this evaluation allowed us to select two materials that are adequate for the biodegradation experiments. Water retention in the solid support materials ranged between 2.5 and 4.5 g H2O g 1 and crude oil recuperation varied between 21.5% and 82.3%. The total nitrogen and total phosphorus content as well as the heterotroph densities were highest in the spent compost compared to the other treatments, while the organic matter content was highest (90.3%) in the sugar cane bagasse waste (Table 1).

M.R. Trejo-Herna´ndez et al. / Chemosphere 68 (2007) 848–855 Table 1 Physicochemical and biological characterization of the support material candidates Parametera

Spent compost

Sugar cane bagasse

Peat moss

Water retention (gH2O g 1 dw) Crude oil recuperation (%) Total nitrogen (mg kg 1) Total phosphorus (mg kg 1) Organic matter (%) Total of heterotrophs (cfu g 1 dw)

2.5

4

4.5

82.3 20 700 3130 50.9 32 · 102

73.4 1800 44 90.3 17 · 102

21.5 1890 196 nd 33

a Per 5 g (dw) of support material; hydrocarbon concentration used: 10 000 (mg kg 1).

Peat moss is a material highly used as support in different processes due to its high water retention ability. However it showed low oil recovery efficiency. In the oil contaminated peat moss, the crude oil recuperation was very low as it forms an emulsion that was difficult to be extracted. Also, this material contains low percentages of N and P. On the basis of this evaluation, the sugar cane bagasse and spent compost wastes were selected for the biodegradation experiments as both materials showed features that are suitable for oil biodegradation process, with the advantage that they contain appreciable densities of autochthonous microorganisms (approximately 102 cfu g 1) that could influence the biodegradation process. In order to identify the best treatment that could ensure sterility of the waste supports, the materials were subjected to several treatments as described earlier. After the treatments the mixed spent compost and sugar cane bagasse wastes (ratio 3:1) were artificially contaminated with 10 000 mg kg 1 of crude oil and incubated for 15 days. All the treatments except mercuric chloride were unable to eliminate the autochthonous microflora present in the reaction mixes as counts in the order of between 102 and 105 cfu g 1 and between 108 and 1010 cfu g 1 were observed at zero and 15 days incubation respectively (Table 2). Also, total petroleum hydrocarbon disappearance rates ranged between 56 and 257.3 mg kg 1 day 1 for all the treatment except mercuric chloride treatment in which no degradation was expectedly observed (Table 2). GC profiles of

Table 2 Changes in microbial density and residual crude oil after sterilization treatment of spent compost and sugar bagasse wastes Treatment

Microbial growth (cfu g 1 dw) Days 0

Without treatment Sodium azide (1%) UV light 5 h exposition Perchloric acid (7%) Mercuric chloride 2%

Total petroleum hydrocarbons (mg kg 1 day 1)

15 4

1 · 10 4 · 105 8 · 102 2 · 102 0

1 · 1010 1 · 108 5 · 109 3 · 109 0

Crude oil concentration used: 10 000 mg kg 1.

56.0 ± 2.0 257.3 ± 8.0 131.3 ± 9.3 162.6 ± 7.3 0

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extracted oils from these treatments corroborate the observed gravimetric measurements, and also suggest that some important transformations of the organic materials occurred (Fig. 1). The gravimetric determination at day zero confirms sorption of hydrocarbons in the altered solid materials by approximately 1.5–5%. However, the GC profiles show evidences that some hydrocarbons fractions were more or less retained in solid matrix. In the bioaugmentation experiments, microbial biomass increased in all cases except in the abiotic control in the order of 108–109 cfu g 1, and crude oil degradation rates varied significantly (P < 0.05) between 8.2 and 180.7 mg kg 1 day 1 (Table 3). To eliminate the contributory effect of the microflora of either waste component in the mixtures, heat sterilization was used before mixing (Table 3). Degradation activity was least and very low in the absence of IMP consortium (presence of wastes autochthonous microflora only). However, among the setups containing IMP consortium, degradation activity was least in the presence of sterilized solid support (90.2 mg kg 1 day 1) even though it contained the highest microbial biomass from IMP consortium (3.0 · 109 cfu g 1), and highest in the presence of solid support with microflora from sugar cane baggase waste only (180.7 mg kg 1 day 1). However, the contributory effect of the autochthonous microflora from either of the waste materials on degradation activity was not comparatively significantly different. 3.2. Kinetics of crude oil biodegradation Considering that the contributory effect of the autochthonous microflora from either of the waste materials on degradation activity was not significantly different, both materials were combined for use in the kinetic studies. In order to optimize biodegradation conditions, preliminary studies were carried out where the effect of air supply rate was evaluated in reactors. In these studies three different aeration rates of 10, 20 and 30 ml min 1 were evaluated. The solid matrix was formed using spent compost and cane bagasse in a 3:1 ratio, 65% moisture and inoculum density of 104–105 cfu g 1 dry weight. Also, abiotic controls were considered to determine volatilization or adsorption losses from texturing materials. Results showed that when aeration rate is increased, biodegradation also increases proportionally, reaching up to 35% increase in oil biodegradation (data not shown) (because the data are from preliminary studies to optimize aeration requirements) not shown because as this was with a 30 ml/min aeration rate, which was then adopted for the kinetic experiments. Microbial growth showed an initial slight increase during the first 5 days from approximately 4.0 · 105 (log10 5.6) cfu g 1 at day zero, followed by a minimal decline for another five days. This decline was probably due to toxic effect on the non hydrocarbon utilizing components of the microflora. The surviving hydrocarbon utilizing communities then increased in density exponentially for another five days to a peak density of approximately

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852

a

After treatment 0 days

After biodegradation 15 days

b

c

d

e

f

Fig. 1. GC-profiles of hydrocarbons on sterilized solid materials at time 0 and 15 days incubation. (a) Without treatment; (b) addition of sodium azide; (c) UV light exposition; (d) addition of perchloric acid; (e) heat treatment and (f) addition of mercuric chloride. The spent compost and bagasse wastes (3:1) were artificially contaminated with 10 000 mg kg 1 of crude oil after sterilization treatment.

4.0 · 109 (log10 9.6) cfu g 1, representing a four order of magnitude increase in microbial biomass, before stagnation of growth (Fig. 2). The biodegradation pattern of the kinetic studies data followed a first order behavior with regular gradual

decreases in residual crude oil to lowest level of about 60% in 15 days after which biodegradation ceased (Fig. 2). Chromatographic analysis of the residual crude oil of the initial and final times of kinetics studies corroborates the observation of the gravimetric estimation showing a significant bio-

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853

Table 3 Effect of bioaugmentation using IMP consortium on microbial growth and biodegradation rates of crude oil Maya in solid state cultures Experiments

Growth biomass (cfu/g dw)

Disappearance rate (mg kg 1 day 1)

1. Inhibited control (addition of HgCl2 2% dw/ww) 2. IMP consortium inoculated on sterilized solid materials wastes 3. IMP consortium inoculated on solid materials wastes with natural microflora 4. Natural microflora from solid wastes 5. IMP consortium inoculated on solid materials wastes with only natural microflora from compost wastesa 6. IMP consortium inoculated on solid materials wastes with only natural microflora from bagasse wastesb

0 3.0 · 109 5.8 · 108 4.7 · 108 4.0 · 108

0.8 ± 0.1 90.2 ± 1.2 123.4 ± 6.5 8.2 ± 1.3 103.3 ± 4.8

7.6 · 108

180.7 ± 8.3

Crude oil concentration: 10 000 mg kg 1; bioaugmentation conditions: IMP consortium density: 1 · 103 cfu g 1 dw; pH 7; initial moisture 60–65%; packed density: 0.9 g ml 1. a Sugar cane bagasse waste component was initially sterilized to eliminate microflora before mixing. b Spent compost component was initially sterilized to eliminate microflora before mixing. All experiments were carried out in triplicate at 28 C.

10

Reduction of crude oil (%))

80 8 60

40 6 20

4

0 0

5

10

15

a

Log10Viable cell density (cfu g-1)

100

20

b

Time (days)

Fig. 2. Crude oil degradation and microbial growth kinetic using solid spent compost and sugar cane bagasse wastes in solid state cultures during 20 days of incubation. Initial crude oil concentration 10 000 mg kg 1.

degradation of the crude oil (Fig. 3). Both gravimetric and gas chromatographic analyses of the abiotic controls revealed that no hydrocarbon loss occurred by volatilization or adsorption of contaminants to the solid support materials (data not shown).

Fig. 3. Chromatographic profile of the residual crude oil at day zero (a) and after 20 days reactions (b) during the kinetic studies.

4. Discussion Hydrocarbon degradation studies carried out with compost showed that compost addition can accelerate hydrocarbon biodegradation processes. First, because of the presence of autochthonous microflora, and second, since oil reduction is increased and little adsorption of hydrocarbons into the support materials were observed. A Previous report has suggested that the presence of autochthonous microflora could have a positive effect on hydrocarbon degradation as the microflora may have the ability to grow in lignocellulosetype substrates and hence promote the degradation of the hydrocarbon pollutant through the action of ligninolytic enzymes which are involved in the oxidation of polyaromatic hydrocarbons (Trejo-Hernandez et al., 2001). Never-

theless, it was demonstrated that hydrocarbons present in soils bind to the organic matter through natural processes of humification (Jones and Tiller, 1999). Previous workers have reported the potentials of these organic materials in the biodegradation of nitro-substituted toluene (Breitung et al., 1996) and polycyclic aromatic hydrocarbon (Ka¨stner et al., 1995), and suggested that the biodegradation was attributed to the presence of native microflora as well as the organic matter they supply. In corroboration with the observation in this study, the potential of the use of organic materials in the acceleration of crude oil biodegradation was highlighted in a previous report (Roldan-Martin et al., 2006) in which low levels of

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orange peels was reported to accelerate biodegradation of petroleum hydrocarbon by as much as 60% in 15 days. Sugar cane bagasse, in particular, have been previously reported to be important reservoirs of microorganisms that can be stimulated for removal of weathered hydrocarbon from contaminated tropical soils (Perez-Armendariz et al., 2004). Also, Barathi and Vasudevan (2003) reported that addition of wheat bran as bulking agent to crude oil contaminated soil rapidly enhanced bioremediation. The spent compost/cane bagasse mix, besides being an important source of N and P nutrients, also provided an appropriate support for the solid matrix as well as native microorganisms that are capable of hydrocarbons utilization, although there is need for acclimatization of the autochthonous microflora to the hydrocarbon as suggested by the lack of biodegradation in the absence of the IMP consortium, in spite of the presence of the microorganisms from spent compost and sugar cane bagasse wastes. Similar observation was reported by Williams et al. (1999) who showed that autochthonous microorganisms from agricultural wastes accelerated degradation of diesel when they were gradually acclimatized. Also, the presence of the exogenous IMP consortium significantly increased biodegradation of the crude oil in line with previous report of Nakles and Loehr (2002) that organic bulking agents allow for successful treatment of higher oil concentrations and increase the biodegradation rate, and this underscores the need for the introduction of exogenous microflora of proven capability for hydrocarbon degradation. Ka¨stner et al. (1995) reported the relevance of exogenous microorganisms from compost wastes for degradation in diesel. Although it is important to ensure that there will be no direct negative interaction amongst the microbial population, but rather a synergistic one that will positively impact on the biodegradation process as suggested by the results of the sterilization experiment (Fig. 1). 5. Conclusions The use of solid wastes in hydrocarbon biodegradation is a promising alternative to soil bio-restoration treatments. Although their effect on hydrocarbon biodegradation is not well known, their porosity which facilitates aeration and water retention makes their use attractive. Also, organic solid wastes such as spent compost and sugar cane bagasse wastes are an additional source of microorganisms and nutrients (N and P) that accelerate hydrocarbon degradation. These materials have been shown to be relevant in the acceleration of crude oil biodegradation not only because of their native microflora, but also due to the enzymatic activities they have been reported elsewhere to possess. Acknowledgements This work was supported by the Mexican Institute of Petroleum (IMP) (Research Grant No. FIES-95-108-VI-

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