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Bioremediation of oil refinery sludge by landfarming in semiarid conditions: Influence on soil microbial activity
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Environmental Research 98 (2005) 185–195 www.elsevier.com/locate/envres
Bioremediation of oil refinery sludge by landfarming in semiarid conditions: Influence on soil microbial activity J.A. Marin, T. Hernandez, C. Garcia Department of Soil and Water Conservation and Waste Management, Centro de Edafologı´a y Biologı´a Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, 30100 Murcia, Spain Received 6 April 2004; received in revised form 2 June 2004; accepted 11 June 2004 Available online 23 July 2004
Abstract Bioremediation of a refinery sludge containing hydrocarbons in a semi-arid climate using landfarming techniques is described. The objective of this study was to assess the ability of this technique to reduce the total hydrocarbon content added to the soil with the refinery sludge in semiarid climate (low rain and high temperature). In addition, we have evaluted the effect of this techique on the microbial activity of the soil involved. For this, biological parameters (carbon fractions, microbial bilmass carbon, basal respiration and ATP) and biochemical parameters(different enzymatic activities) were determined. The results showed that 80% of the hydrocarbons were eiminated in eleven months, half of this reduction taking place during the first three months. The labile carbon fractions, MBC, basal respiration and ATP of the soils submitted to landfarming showed higher values than the control soil during the first months of the process, although these values fell down by the end of the experimental period as the hydrocarbons were degraded by mineralisation. All the enzymatic activities studied: oxydoreductases such as dehydrogenase activity, and hydrolases of C(b-glucosidase activity) and N Cycle (urease and protease) showed higher values in the soils amended with the refinery sludge than in the control. As in the case of the previous parameters, these value fell down as the bioremediation of the hydrocarbons progressed, many of them reaching levels similar to those of the control soil after eleven months. r 2004 Elsevier Inc. All rights reserved. Keywords: Bioremediation; Landfarming; Refinery sludge; Microbial activity; Semiarid conditions
1. Introduction The petrochemical industry generates a series of liquid effluents during the petroleum-refining process. These effluents must be treated through depuration processes. The sludges (oil refinery sludges) that result from this depuration process have a high content of petroleumderived hydrocarbons, mainly alkanes and paraffin of 1–40 carbon atoms, along with cycloalkanes and aromatic compounds (Overcash and Pal, 1979); thus it is a potentially dangerous waste product. Simply dumping these wastes or burning them with no previous treatment has serious environmental consequences and presents a risk to both ecosystems and human health (Baheri and Meysami, 2001). Corresponding author. Fax: +34-968396213.
E-mail address: [email protected] (C. Garcia). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.06.005
Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be eliminated from the environment (Leahy and Colwell, 1990). Landfarming is a frequently chosen treatment method for petroleum hydrocarbon-contaminated soils because of containment, relatively low cost, and high potential for success (Harmsen, 1991). The landfarming of pertroleum hydrocarbon wastes and criteria for designing, operations, and monitoring landfarming have been addressed in detail by Kincannon (1972) and Sims et al. (1989). Such landfarming involves the use of the natural biological, chemical, and physical processes in the petroleum-contaminated soil to transform the organic contaminants of concern (Pope and Matthews, 1993). Biological activity apparently accounts for most of the transformation of organic
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contaminants in soil, although physical and chemical mechanisms may provide significant pathways for some compounds. The landfarming concept serves as a basis for the design and operation of soil bioremediation technologies in a large number of waste sites requiring cleanup (Pope and Matthews, 1993). The importance of microbial activity in cycling organic matter and regulating active nutrient pools in soils suggests that the effects of pollution on soil microorganisms are fundamentally related to the effects on crops, natural vegetation, and ecosystem productivity (Ladd et al., 1996). Microbial activity measurements therefore appear as good indicators of the degree of pollution of contaminated soils (Insam et al., 1996; Kuperman and Margaret, 1997). Nannipieri et al. (1990) showed that the simultaneous measurement of the activity of several enzymes in soil may be more valid for estimating the overall microbial activity and its response to diffuse pollution and environmental stress than the determination of the activity of a single enzyme. It also gives information on the diversity of functions that can be assumed by the microorganisms in soils, which is one of the main questions in the field of sustainable soil management (Burns, 1982; Kennedy and Smith, 1995). Microbial activity is in fact a general term that includes all the metabolic reactions and interactions conducted by the microflora and microfauna in soil (Nannipieri et al., 1990). Soil properties that are important in influencing the rate and extent of the remediation of petroleumcontaminated soils include texture, bulk density, hydraulic conductivity, CEC, nutrient status, and soil microorganisms type and numbers (Atlas, 1981; Pope and Matthews, 1993; Sims et al., 1989). However, parameters indicative of metabolic activity (biological and biochemical parameters) have been scarcely studied in soils with waste petroleum subjected to landfarming. The main aim of this study was to evaluate the ability of landfarming as an useful technique for eliminating total hydracarbon added to soil with refinery sludge, particularly when semiarid conditions (low rain, high temperature) are stabilized. In addition, this study investigated the evolution of the microbial activity when landfarming is used, to support the use of these microbial assays as bioindicators of biodegradation and/or for concern about soil quality after bioremediation.
2. Materials and methods 2.1. Organic material The organic material consisted of a sludge resulting from the depuration of effluents from an oil refinery. According to Oolman et al. (1992), our sludge generated in refineries was submitted to a process of centrifugation to eliminate some of the water, giving them a semisolid
Table 1 Oil refinery sludge characteristics (dry weight) Total organic carbon (g kg1 ) Chloride (g kg1 ) Sulfates (g kg1 ) Nitrites (mg kg1 ) Fenols (mg kg1 ) Fluor (mg kg1 ) Ammonia (mg kg1 ) Chloride solvents (mg kg1 ) As (mg kg1 ) Pb (mg kg1 ) Hg (mg kg1 ) Cd (mg kg1 ) Ni (mg kg1 ) Zn (mg kg1 ) Cu (mg kg1 ) Cr (mg kg1 ) Hydrocarbon content (g kg1 )
162.3 5.6 24.2 3.90 377 o1 12.15 o1 o5:04 10.1 o1:58 o1:2 24 57 22 21 220.7
Table 2 Soil characteristics pH (1:2.5) EC(1:5) ðmS cm1 Þ Available K (meq 100 g1 ) Available Ca (meq 100 g1 ) Available Mg (meq 100 g1 ) Available Na (meq 100 g1 )
7.35 299 0.49 10.77 2.40 0.23
Total organic matter (g kg1 ) Total organic carbon (g kg1 ) Total nitrogen (g kg1 ) C/N Total carbonates (g kg1 ) Active limestone (g kg1 ) Available (P mg kg1 ) Chloride (meq 100 g1 ) Sulfates (meq 100 g1 ) Hydrocarbon content (g kg1 ) Available (Fe mg kg1 ) Available (Cu mg kg1 ) Available (Mn mg kg1 ) Available (Zn mg kg1 )
8.7 5.1 3.7 1.38 151.6 72.7 18.72 0.79 0.26 6.0 29.37 4.97 5.65 18.81
consistency (700–800 g kg1 moisture). The principal chemical–physical characteristics of the sludge used in our experiment are summarized in Table 1. The soil in which the experiment was carried out, which was very near the refinery that produced the sludges used, is poor in organic matter and nutrients and has a metal content that reflects its proximity to a mining area (Table 2). 2.2. Site, landfarming process, and sampling procedure The study was conducted in Cantagena, Murcia (SE Spain), an area with a predominantly semiarid climate.
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The annual mean temperature is 19 C and total rainfall ranges 200–250 mm per year, mostly concentrated in autumn and spring. The soil used in the experiment is typical of soils found in SE Spain (see Table 2). The clayey subsoil confers a degree of impermeability, which makes it suitable for recycling refinery sludges through landfarming. Three 600-m2 plots for carrying out the bioremediation process (landfarming) and another three for control soil were established; plots were surrounded by fences to prevent animals from straying onto them. The amount of waste residue to mix with the topsoil depends on the degradation rate, structure, and texture of the residue (Hillel, 1989). These factors, together with the amount of rainfall determine the time and space for retaining and degrading the contaminant. In an evaluation by Symons and Sims (1988), the extent and rate of detoxification of petroleum-contaminated soils undergoing landfarming treatment were directly related to waste loading rate. A previous study of the same soil in field conditions (Marin Milla´n, 2004) revealed that an initial application of 5–6% w/w total hydrocarbon content (TPH) on the topsoil showed the best result for degradation; thus, this was the leading rate applied in our landfarming experiment. Landfarming consisted of depositing the refinery sludge on the soil surface and mixing it with the top 1 m of the soil 1 week later by means of a tractor. Subsequent treatment simply consisted of aerating the top 1 m of soil once a month with a tractor. Control plots were treated in the same way as landfarming plots. Conditions were totally natural with no added water or nutrients; the minimum aeration was to ensure that the bioremediation experiment was as cheap as possible to run. At different times after adding the sludge (1,3,5,7,9,11 months) samples were taken from each of the plots. In the experimental plots, the sample was taken before each monthly aeration. Composite soil samples of the top 15 cm were randomly collected from six different places in each site. Each soil sample consisted of six 150-cm3 subsamples, which were thoroughly mixed to obtain a composite sample. The samples were brought to the laboratory on the same day and kept in the refrigerator at 4 C until they were analyzed (microbiological analysis were carried out 3–4 days after sampling). 2.3. Chemical parameters Electrical conductivity (EC) and pH were measured in a 1/5 solid/liquid aqueous extract. Total organic carbon was measured by the Bakley–Black method. In the oil sludge, chloride, nitrites, sulfate, ammonium, and fluor were measured by HPLC. TPH was measured by the infrared USEPA method (1983). Water-soluble carbon was measured in the extract 1/10 (solid/liquid) in automatic total organic
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carbon (TOC) analyzer. Water-soluble carbohydrates, in the same extract, were measured with anthrone by the Brink et al. (1960) method and soluble polyphenol compounds by the Kuwatsuka and Shindo (1973) method. 2.4. Microbiological analysis Microbial biomass carbon (MBC) was determined using a fumigation–extraction procedure (Vance et al., 1987). The 0.5 M K2 SO4 -extracted C was measured as indicated for water-soluble C. Basal respiration was determined in 50 g dry soil placed in hermetically sealed flasks, moistened to 50% of its water-holding capacity, and incubated in the dark at 28 C. The CO2 released was measured by a IR CO2 detector. The metabolic quotient ðqCO2 Þ was calculated by dividing the C–CO2 released from the sample in 1 h by the microbial biomass carbon content. The microbial biomass carbon to total organic carbon ratio (MBC/TOC) was also calculated. 2.5. Enzymatic assays Dehydrogenase activity. (DHA): Dehydrogenase activity was determined by the Skujins (1976) method as modified by Garcia et al. (1997). Soil ð1 gÞ at 60% of its field water-holding capacity was treated with 0:2 mL of 0.4% 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride in distilled water for 20 h at 22 C in darkness. The iodo-nitrotetrazolium formazan formed was extracted with 10 mL of a mixture of 1:1.5 ethylene/ chloride acetone by shaking vigorously for 1 min and filtering through a Whatman No. 5 filter paper. Iodonitrotetrazolium formazan was measured spectrophotometrically at 490 nm. Urease activity: Two milliliters of phosphate buffer (pH 7) and 0:5 mL of 6.4% urea were added to 0:5 g of soil and then the mixture was incubated at 30 C for 90 min before the volume was made up to 10 mL with distilled water. The ammonium released after addition of 0:1 mL 10 M NaOH was measured using an ammonium-selective electrode (CRISON micro pH 2002). A control without urea was used with each sample (Nannipieri et al., 1980). Protease activity on N-a benzoyl-L-argininamide (protease-BAA): Two milliliters of phosphate buffer (pH 7) and 0:5 mL of 0.03 M N-a-benzoyl-L-argininamide (BAA) substrate were added to 0:5 g of soil. The mixture was incubated at 37 C for 90 min and then diluted up to 10 mL with distilled water. The ammonium released was measured in the same way as that for urease (Nannipieri et al., 1980). Phosphatase activity: Two milliliters of 0.1 M maleate buffer (pH 6.5) and 0:5 mL of 0.115 M p-nitrophenyl phosphate were added to 0:5 g of soil and incubated at 37 C for 90 min. the reaction was stopped by cooling at
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2 C for 15 min and then 0:5 mL of 0.5 M CaCl2 and 2 ml of 0.5 M NaOH were added to the soil mixture before centrifugation at 4000 rpm for 5 min. The pnitrophenol formed was determined in a spectrophotometer at 398 nm (Tabatabai and Bremner, 1969). Controls were made in the same way, although the substrate was added before the CaCl2 and NaOH. b-Glucosidase activity: Two milliliters of 0.1 M maleate buffer (pH 6.5) and 0:5 mL of 50 M pnitrophenyl-b-D-glucopyranoside were added to 0:5 g of soil. Then the same procedure reported for phosphatase assay was follwed, except that the NaOH was substituted by Tris buffer, pH 12.
2.6. Ecotoxicity assay A toxicity test was carried out using luminescent bacteria (microtox), in which the inhibition of the luminescence of Photobacterium phosphoreum was measured using a luminometer (Kapanen and Ita¨vaara, 2001) after adding extracts of the samples. The microtox assay has been used to evaluate the rate and extent of detoxification of petroleum-contaminated soils under landfarming conditions (Symons and Sims, 1988). This assay uses a suspension of luminescent bacteria (P. phosphoreum) as bioassay organism for measuring acute toxicity in aqueous extracts of soil (Bulich, 1979; Matthews and Hastings, 1987). Lyophilized bacteria were used after rehydration in the commercial solution. All assays were carried out at 15 C with 15- and 30-min contact periods between 0:5 mL of bacterial suspension and soil suspension. Soil suspension was prepared by mixing 1 g soil with 10 mL of 2% NaCl (w/w) solution.
3. Results and discussion 3.1. Refinery sludge The main nonmetal compounds contained in the refinery sludge used in the experiment were organic compounds (hydrocarbons) (Table 1), especially alkanes, paraffins, aromatics, phenols, asphaltenes, and polynuclear aromatic hydrocarbons (Concawe, 1980). As is known, some of these compounds can be considered labile because they are easily degraded by microorganisms (simple aliphatic and aromatic compounds), while heavier components (such as asphaltenes) are more recalcitrant and difficult to degrade by bioremediation (Marin Milla´n, 2004). An important point is the high chloride content in the refinery sludge (Table 1), which is responsible for the high electrical conductivity values of the material, which means that great care should be taken if it is used for soil disposal as the excessive salt content may inhibit soil microbial activity (Garcia and Herna´ndez, 1996). Analysis of the heavy metal content of the semisolid refinery sludge indicated high quantities of Zn, Pb, and Ni. However, we must accept that the accumulation of such metals following the addition of refinery sludge does not necessarily have a negative effect on the microbial populations which carry out the hydrocarbon degradation processes, since studies on ecological doses in heavy-metal-contaminated soils have shown that very high levels of soluble metals are needed to negatively influence the soil’s metabolic activity (Moreno et al., 2003). More problematic in this sense can be the quantity of phenols found in the sludge, since they may well have a negative effect on the microbial population responsible for hydrocarbon degradation (Concawe, 1980), or even salinity, which may, as we have already pointed, alter such microbial activity.
2.7. Statistical analysis 3.2. pH and electrical conductivity in landforming soils Statistical analysis (ANOVA analysis and LSD test for mean) was performed with the Statgraph program on data obtained in the control and landfarming plots.
The bioremediation of hydrocarbons of the refinery sludge will be carried out by soil microorganisms; so soil
Table 3 pH and electrical conductivity (EC) in the landfarming and control plots Time elapsed after oil sludge addition (months)
EC ðdS m1 Þ
pH Control plots
Landfarming
Control plots
Landfarming
1 3 5 7 9 11
7.70 8.24 8.68 8.07 8.62 8.68
7.59 7.57 7.50 7.56 7.61 7.65
0.90 0.49 0.60 1.09 0.40 0.27
3.38 3.48 2.12 2.20 2.79 2.71
LSD ðPo0:05Þ
0:51
0:32
0:35
0:45
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characteristics that influence soil microbial activity such as pH will also influence hydrocarbon degradation. Most heterotrophic bacteria and fungi need a pH near neutrality, with fungi being more tolerant of acidic conditions (Atlas, 1988). Extreme pH values could have a negative effect on the ability of microbial populations to degrade hydrocarbons. Dibble and Bartha (1979) observed an optimal pH of 7.8 for the mineralization of oily sludge in soil. In our case, the pH values of the landfarming plot ranged between 7.5 and 7.65, a range suitable for microbial growth (Table 3). Soils with oil sewage showed higher EC values than control soils (Table 3). There are few published studies dealing with the effects of salinity on the microbial degradation of hydrocarbons. Shiaris (1989) reported a general positive correlation between the salinity and the rates of mineralization of phenanthrene of naphtalene. Kerr and Capone (1988) observed a relationship between the mineralization rate and the salinity in sediments of the Hudson river. In contrast, Ward and Brock (1978) showed that rates of hydrocarbon metabolism decreased with increasing salinity in the range 3.3–28.4% and attributed the results to a general reduction in microbial metabolic rates. In our case, and according to previous studies (Garcia and Herna´ndez, 1996), the salinity detected in the landfarming plots (Table 3) could have a negative effect on the microbial activity in these soils and so on hydrocarbon bioremediation, especially when the salinity is due to chlorides as opposed to sulfates.
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3.3. Carbon fractions in landfarming soils Fig. 1 shows that the content of total organic carbon at the beginning of the experiment was greater in the landfarming than in the control plots, although these levels decreased by 27% during the experiment. It was to be expected that carbon compounds would be degraded to a greater extent during the first months of landfarming because of the aeration carried out which should favor the mineralization of the organic matter and provoke the degradation of the hydrocarbons contained in the refinery sludge. It is possible that the technique used to measure the TOC (Wakley–Black method) produces an error, especially at the outset, when there are more labile hydrocarbons that may be affected by the temperature reached with this method when soil samples are treated with sulfuric acid. The most labile fractions of C (polyphenolic compounds and water-soluble carbohydrates and carbon) constitute some of the most immediate energy sources of microorganisms and their levels reflect microbial activity in general (Garcia et al., 2000). These fractions are continually formed and degraded due to the mineralization of the organic matter and microbial synthesis. The addition of refinery sludge increased the proportion of these labile C fractions compared with that found in the control soil (Fig. 1). The behavior of these carbonaceous fractions in the landfarming plots was similar, the high values at the outset diminishing since they were constantly used as energy source by the microorganisms. The successive aeration would have contributed to the
Fig. 1. Content of different carbon fractions in the landfarming and control plots.
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mineralization of the carbon compounds, as has been confirmed by several authors in studies of turning agricultural soils (Haynes and Tregurtha, 1999; Hussain et al., 1999). In the last stages of landfarming these fractions tended to be partially regenerated, perhaps because of the decomposition of other, more recalcitrant carbon molecules. This could explain why, at the end of the experiment, the values obtained for the labile carbon fractions exceeded those of the control soils. In the case of the polyphenols, their disappearance rate was slower than those of the water-soluble carbon and watersoluble carbohydrates and it took 7 months to reach their minimum level since their more complex structure makes them more resistant to biodegradation. This is not a positive finding since some studies (Concawe, 1980) mention that a high concentration of phenols in a soil has a negative effect on the microbiol populations capable of degrading hydrocarbons.
3.4. Hydrocarbons content in landfarming soils Fig. 2 shows the soil hydrocarbon content as a function of time. It is clear that the bioremediation method followed (simple aeration of the soil with the sludge) significantly reduces the hydrocarbon content of the sludge (80% reduction in 11 months). Aliphatic and aromatic hydrocarbons are degraded in the highest quantity according to Marin Milla´n (2004). The initial steps in the catabolism of aliphatic, cycling, and aromatic hydrocarbons by bacteria and fungi involve the oxidation of the substrate by oxygenases, for which molecular oxygen is required. Aerobic conditions are therefore necessary for this route of microbial oxidation of hydrocarbons in the environment. Under semiarid conditions with scarce rain, the biodegradation of hydrocarbons has taken place in 11 months. This time could probably be shorter if soil humidity (in our case,
Fig. 2. Hydrocarbons content of the soil during landfarming experiment (LSD, Po0:05 : 0:12 for landfarming plots and 0.08 for control soil).
1–3%) is higher, but it is not possible under semiarid conditions. The loss of organic compounds in landfarming plots is governed by a first-order kinetics, the elimination rate of a compound being proportional to its concentration (Demque et al., 1997). During the degradation process two clearly differentiated phases were observed: a first stage with a high velocity, in which the hydrocarbon degradation rate was maximal (55% of the total hydrocarbon degradation takes place in 2 months, from month 1 to month 3) and the most labile fractions (mainly aliphatic) were involved, and a second slower stage (45% of the total hydrocarbon degradation takes place in 8 months, from month 3 to month 11). The first stage lasted 2 months, after which the degradation rate slowed down, demonstrating that, as the most easily biodegradable hydrocarbons are consumed, the microorganisms turn their attention to other fractions, such as the aromatic, condensed cyloalkanes, etc., which are degraded at different rates (Langbehn and Steinhart, 1995). After 9 months the degradation rate slowed down sharply, since the remaining fractions are structurally more complex hydrocarbons and therefore less accessible, their recalcitrance and low bioavailability causing the activity of the microbial populations to drop (Margesin et al., 1999). Sims and Sims(1999) also mentioned the biphasic kinetics of petroleum hydrocarbons during degradation by landfarming. This degradation rate depends on, among other factors, the ability to control environmental variables. For example, temperature influences petroleum biodegradation by its effect on the physical nature and chemical composition of the oil, rate of hydrocarbon metabolism by microorganisms, and the composition of the microbial community (Atlas, 1981). 3.5. Metabolic activity in landfarming soils In a soil where a landfarming process is being carried out (for example, for reducing its hydrocarbon content as in our case), the microbial population and its activity could be influenced for that process (quantity of toxic in the soil); therefore, the study of some parameters indicative of microbial activity can be of interest. The microbial biomass carbon includes only 0.5–4.6% of the soil TOC content but is responsible for the decomposition of elements involved in soil fertility (Dı´ az-Ravin˜a et al., 1993). MBC has a turnover rate that is several times faster than that of organic matter and so the flow of nutrients from this fraction is greater than that from others, which explains its influence on the availability of nutrients to plants (Sparling, 1992). In our experiment the landfarming plots showed MBC values that were considerably above those of the control soils (Fig. 3), demonstrating that microbial biomass size and activity increased with the incorporation of the sludge. At the
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seemed to be closely correlated with that of MBC during the landfarming process. The ATP content was also higher at all times in the soil with refinery sludge than in the control soil, especially during the first months, when the rate of metabolic activity was at its highest. During this time the microorganisms that are responsible for degrading the hydrocarbons set to work and increase in numbers. These microbial populations diminish in size as the substrates are used up and mineralized, the ATP content decreasing since it can exist only inside living cells. Soil respiration measurements give an idea of the microbial activity in the soil and of the quantity and quality of substrates susceptible to mineralization (Anderson, 1982). In our experiment, the microbial activity was high just after addition of the refinery sludge since the most labile hydrocarbon compounds present need high microbial activity if they are to be degraded (Fig. 3); hence, the increase in respiration recorded. CO2 emission gradually decreases as the most labile hydrocarbon fractions disappear, with only the most recalcitrant fractions remaining. However, such behavior is very susceptible to humidity and temperature (Leiros et al., 1999). Humidity, for example, conditions the capacity of microorganisms to mineralize organic matter, which is why long periods of drought like those occurring in the study area decrease soil microbial activity (Orchard and Cook, 1983). Temperature has both a direct and an indirect effect on such processes. (Davidson et al., 1998). In our case, the minimum temperature were never low enough to stop respiration, although the maximum temperatures, for their indirect effect on drought, could be said to have a negative influence. All parameters studied indicate that microbial activity diminishes with the bioremediation process but is always higher than that of the control soil. 3.6. Metabolic quotient and MBC/TOC ratio100 Fig. 3. Microbiological parameters in the landfarming and control plots.
beginning of the experiment some sort of inhibition that affects MBC seems to exist; this is not unexpected given the large quantity of exogeneous organic matter and high amount of hydrocarbon added. It is known that this parameter is subject to seasonal variations (Nannipieri et al., 1990), although we think that any variation in this sense is much less than the influence of the addition of the refinery sludge. Nannipieri et al. (2002) also indicated that climates with wide thermal variations (such as dry tropical) seemed to have the greatest influence on seasonal variations of microbial biomass. Another parameter that can be used to measure the overall microbial activity of a soil is the adenosin-5triphosphate (ATP) content (Fig. 3), whose behavior
The ðqCO2 Þ reflects the efficiency with which the carbon is used. This parameter can discriminate between the maturity levels of ecosystems since it is assumed that mature systems respire less per unit of biomass because they canalize less energy toward their metabolism. It can also act as an index of stress in the face of contamination (Wardle and Ghani, 1995; Insam and Domsch, 1988; Anderson and Domsch, 1993). The values of qCO2 increased very significantly in the landfarming plots after the addition of the sludge (Table 4), which agrees with the observations of other authors such as Marin Millan (2004), who suggested that a disturbance (as the addition of sludge in our experiment might be interpreted) can increase the metabolic coefficient. The reason for this in our case would be that microorganisms must raise their respiration per unit of biomass to
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Table 4 Metabolic quotient (qCO2), MBC=TOC 100 ratio, and dehydrogenase activity (DHA) in the landfarming and control plots Time elapsed after sludge oil addition (months)
1 3 5 7 9 11 MDS Po0:05
qCO2(mg C–CO2mg MBC g1 h1 )
MBC=TOC 100
Control
Landfarming
Control
Landfarming
Control
Landfarming
6.6 4.1 2.3 5.1 2.8 4.2 2:3
1.36 8.7 6.6 9.9 8.3 11.9 2:9
0.56 1.08 0.44 0.23 0.84 1.43 0:34
7.29 5.15 2.42 1.46 1.29 1.77 0:45
73.0 32.0 70.7 35.4 43.1 38.9 10:3
197.1 156.8 99.4 90.6 89.0 62.6 21:5
degrade the hydrocarbons. At the end of the experiment, the qCO2 values did not fall, perhaps because the system was not reaching a certain degreee of equilibrium after the disappearance of the easily biodegradable hydrocarbons during the first months. The MBC/TOC ratio was higher in the soils receiving the sludge than in the control soils (Table 4). It is clear that the microbial biomass increases in quantity to help in the biodegradation of substrates such as hydrocarbons. At the end of the landfarming process, when most of the easily biodegradable hydrocarbons present in the refinery sludge have been degraded by the microorganisms existing in the soil, the ratio decreased. The existence of a minor quantity of available substrates for microorganisms could be responsible for this result. 3.7. Enzyme activities Soil enzymes are biological catalysts of specific reactions and these reactions, in turn, depend on a variety of factors. Numerous studies of enzymes exist and comparisons have been made in various soils subjected to different climatic conditions and management practices (Burns, 1978; Ladd, 1985). Nonpolar organic compounds, such as hydrocarbons, are hydrophobic and do not interact significantly with proteins in solution. In soil, crude oil at high concentrations ð100 kg m2 Þ may block the expression of enzyme activity by coating organomineral and cell surfaces and thereby prevent soluble substrates from reaching the enzyme molecules (Speir and Ross, 2002). These authors indicate that, at moderate levels of hydrocarbon contamination, some enzyme activities declined and some increased. Dehydrogenase activity has been used to assess microbial activity, although this use has been criticized by different authors; Benefield et al. (1977) indicated that DHA is not an accurate parameter for determining the electron flow rate to O2 because the electron acceptors used in the dehydrogenase assays are less efficient than O2. However, Garcia et al. (1997) found that DHA is a good index of the status of soil microbial
DHA (ng INTF g1 )
activity in semiarid Mediterranean areas. Our data confirm that DHA is higher in landfarming plots than control soil probably due to higher microbial activity in these plots. DHA has been widely used to measure the catabolic activities in soil, which are correlated with microbial activity (Skujins, 1976). In the landfarming plots, DHA diminishes with the time; when hydrocarbons have already been degraded, microbial activity decreases and DHA is lower. The hydrolases belonging to the cycles of elements such as nitrogen (urease and protease activity which hydrolyse BAA), phosphorus (phosphatase activity), or carbon (b-glucosidase activity) were determined in the control soil and in those with the refinery sludge subjected to landfarming (Fig. 4). In the first 3–5 months the activity in the landfarming soils was higher than that in the corresponding controls. As Rastin et al. (1998) pointed out, enzymatic activities are influenced by the biological matter of the soil, in our case by the addition of the refinery sludge and the changes in the soil as a result of landfarming. It is clear that the cycles of the most important elements in the soil are affected by the introduction of the refinery sludges. After the most labile hydrocarbons of the sludge have been degraded, the enzymatic activities return to values similar to those of the control soil (Fig. 4).
3.8. Ecotoxicity with luminiscent bacteria This toxicity assay provides information complementary to the microbial activity measurements described above. In fact, bioassays are generally used to check for toxic elements that are bioavailable for exogenous organisms (Ro¨nnpagel et al., 1998). Concawe (1980) summarized the environmental toxicological effects of petroleum refinery effluents and found that, in general, oils increase in toxicity with increasing levels of lowboiling compounds, unsaturated compounds, and aromatics. Also aromatics with increased numbers of alkyl substituents have higher toxicity, and toxicity increases along the series alkanes–alkenes–aromatics.
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Fig. 4. Enzyme activities in the landfarming and control plots.
Fig. 5. Percentage of inhibition on luminescent bacteria during landfarming experiment (LSD, Po0:05 : 14:5 for landfarming plots and 2:1 for control soil).
Cycloalkanes and cycloalkenes appear to be more toxic than alkanes. Fig. 5 represents the percentage of the inhibition of luminescent bacteria. As can be appreciated, the level of inhibition decreased dramatically in the soils subjected to landfarming. As the extracts treated with this method are aqueous extracts, these findings indicate that the water-soluble toxic compounds are totally sanitized after 9 months.
conditions. The biodegradation of total hydrocarbons has been established at 80% in 11 months. Dry conditions in semiarid climate, where the rain is very scarce, can be responsible for this behavior. In general, parameters indicative of soil microbial activity measured during the process of hydrocarbon degradation showed higher values than the control soil; nonpolar organic compounds (as hydrocarbon content in refinery sludge) are hydrophobic and do not interact significantly with proteins in solution, and for this reason enzyme activities are not particularly inhibited under these conditions. After the most labile hydrocarbons of the sludge have been degraded, the enzymatic activities diminish.
Acknowledgements The research was financially supported by RepsolYPF (Research Contract between Repsol-YPF and CEBAS-CSIC). The authors thank Mr. Jesus Mellado, Mr. Francisco Navarro, and Mr. Angel Crespo for their help with the manuscript.
References 4. Conclusion In conclusion, we can stay that the bioremediation of refinery sludge by landfarming is possible is semiarid
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Environmental Research 98 (2005) 185–195 www.elsevier.com/locate/envres
Bioremediation of oil refinery sludge by landfarming in semiarid conditions: Influence on soil microbial activity J.A. Marin, T. Hernandez, C. Garcia Department of Soil and Water Conservation and Waste Management, Centro de Edafologı´a y Biologı´a Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, 30100 Murcia, Spain Received 6 April 2004; received in revised form 2 June 2004; accepted 11 June 2004 Available online 23 July 2004
Abstract Bioremediation of a refinery sludge containing hydrocarbons in a semi-arid climate using landfarming techniques is described. The objective of this study was to assess the ability of this technique to reduce the total hydrocarbon content added to the soil with the refinery sludge in semiarid climate (low rain and high temperature). In addition, we have evaluted the effect of this techique on the microbial activity of the soil involved. For this, biological parameters (carbon fractions, microbial bilmass carbon, basal respiration and ATP) and biochemical parameters(different enzymatic activities) were determined. The results showed that 80% of the hydrocarbons were eiminated in eleven months, half of this reduction taking place during the first three months. The labile carbon fractions, MBC, basal respiration and ATP of the soils submitted to landfarming showed higher values than the control soil during the first months of the process, although these values fell down by the end of the experimental period as the hydrocarbons were degraded by mineralisation. All the enzymatic activities studied: oxydoreductases such as dehydrogenase activity, and hydrolases of C(b-glucosidase activity) and N Cycle (urease and protease) showed higher values in the soils amended with the refinery sludge than in the control. As in the case of the previous parameters, these value fell down as the bioremediation of the hydrocarbons progressed, many of them reaching levels similar to those of the control soil after eleven months. r 2004 Elsevier Inc. All rights reserved. Keywords: Bioremediation; Landfarming; Refinery sludge; Microbial activity; Semiarid conditions
1. Introduction The petrochemical industry generates a series of liquid effluents during the petroleum-refining process. These effluents must be treated through depuration processes. The sludges (oil refinery sludges) that result from this depuration process have a high content of petroleumderived hydrocarbons, mainly alkanes and paraffin of 1–40 carbon atoms, along with cycloalkanes and aromatic compounds (Overcash and Pal, 1979); thus it is a potentially dangerous waste product. Simply dumping these wastes or burning them with no previous treatment has serious environmental consequences and presents a risk to both ecosystems and human health (Baheri and Meysami, 2001). Corresponding author. Fax: +34-968396213.
E-mail address: [email protected] (C. Garcia). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.06.005
Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be eliminated from the environment (Leahy and Colwell, 1990). Landfarming is a frequently chosen treatment method for petroleum hydrocarbon-contaminated soils because of containment, relatively low cost, and high potential for success (Harmsen, 1991). The landfarming of pertroleum hydrocarbon wastes and criteria for designing, operations, and monitoring landfarming have been addressed in detail by Kincannon (1972) and Sims et al. (1989). Such landfarming involves the use of the natural biological, chemical, and physical processes in the petroleum-contaminated soil to transform the organic contaminants of concern (Pope and Matthews, 1993). Biological activity apparently accounts for most of the transformation of organic
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contaminants in soil, although physical and chemical mechanisms may provide significant pathways for some compounds. The landfarming concept serves as a basis for the design and operation of soil bioremediation technologies in a large number of waste sites requiring cleanup (Pope and Matthews, 1993). The importance of microbial activity in cycling organic matter and regulating active nutrient pools in soils suggests that the effects of pollution on soil microorganisms are fundamentally related to the effects on crops, natural vegetation, and ecosystem productivity (Ladd et al., 1996). Microbial activity measurements therefore appear as good indicators of the degree of pollution of contaminated soils (Insam et al., 1996; Kuperman and Margaret, 1997). Nannipieri et al. (1990) showed that the simultaneous measurement of the activity of several enzymes in soil may be more valid for estimating the overall microbial activity and its response to diffuse pollution and environmental stress than the determination of the activity of a single enzyme. It also gives information on the diversity of functions that can be assumed by the microorganisms in soils, which is one of the main questions in the field of sustainable soil management (Burns, 1982; Kennedy and Smith, 1995). Microbial activity is in fact a general term that includes all the metabolic reactions and interactions conducted by the microflora and microfauna in soil (Nannipieri et al., 1990). Soil properties that are important in influencing the rate and extent of the remediation of petroleumcontaminated soils include texture, bulk density, hydraulic conductivity, CEC, nutrient status, and soil microorganisms type and numbers (Atlas, 1981; Pope and Matthews, 1993; Sims et al., 1989). However, parameters indicative of metabolic activity (biological and biochemical parameters) have been scarcely studied in soils with waste petroleum subjected to landfarming. The main aim of this study was to evaluate the ability of landfarming as an useful technique for eliminating total hydracarbon added to soil with refinery sludge, particularly when semiarid conditions (low rain, high temperature) are stabilized. In addition, this study investigated the evolution of the microbial activity when landfarming is used, to support the use of these microbial assays as bioindicators of biodegradation and/or for concern about soil quality after bioremediation.
2. Materials and methods 2.1. Organic material The organic material consisted of a sludge resulting from the depuration of effluents from an oil refinery. According to Oolman et al. (1992), our sludge generated in refineries was submitted to a process of centrifugation to eliminate some of the water, giving them a semisolid
Table 1 Oil refinery sludge characteristics (dry weight) Total organic carbon (g kg1 ) Chloride (g kg1 ) Sulfates (g kg1 ) Nitrites (mg kg1 ) Fenols (mg kg1 ) Fluor (mg kg1 ) Ammonia (mg kg1 ) Chloride solvents (mg kg1 ) As (mg kg1 ) Pb (mg kg1 ) Hg (mg kg1 ) Cd (mg kg1 ) Ni (mg kg1 ) Zn (mg kg1 ) Cu (mg kg1 ) Cr (mg kg1 ) Hydrocarbon content (g kg1 )
162.3 5.6 24.2 3.90 377 o1 12.15 o1 o5:04 10.1 o1:58 o1:2 24 57 22 21 220.7
Table 2 Soil characteristics pH (1:2.5) EC(1:5) ðmS cm1 Þ Available K (meq 100 g1 ) Available Ca (meq 100 g1 ) Available Mg (meq 100 g1 ) Available Na (meq 100 g1 )
7.35 299 0.49 10.77 2.40 0.23
Total organic matter (g kg1 ) Total organic carbon (g kg1 ) Total nitrogen (g kg1 ) C/N Total carbonates (g kg1 ) Active limestone (g kg1 ) Available (P mg kg1 ) Chloride (meq 100 g1 ) Sulfates (meq 100 g1 ) Hydrocarbon content (g kg1 ) Available (Fe mg kg1 ) Available (Cu mg kg1 ) Available (Mn mg kg1 ) Available (Zn mg kg1 )
8.7 5.1 3.7 1.38 151.6 72.7 18.72 0.79 0.26 6.0 29.37 4.97 5.65 18.81
consistency (700–800 g kg1 moisture). The principal chemical–physical characteristics of the sludge used in our experiment are summarized in Table 1. The soil in which the experiment was carried out, which was very near the refinery that produced the sludges used, is poor in organic matter and nutrients and has a metal content that reflects its proximity to a mining area (Table 2). 2.2. Site, landfarming process, and sampling procedure The study was conducted in Cantagena, Murcia (SE Spain), an area with a predominantly semiarid climate.
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The annual mean temperature is 19 C and total rainfall ranges 200–250 mm per year, mostly concentrated in autumn and spring. The soil used in the experiment is typical of soils found in SE Spain (see Table 2). The clayey subsoil confers a degree of impermeability, which makes it suitable for recycling refinery sludges through landfarming. Three 600-m2 plots for carrying out the bioremediation process (landfarming) and another three for control soil were established; plots were surrounded by fences to prevent animals from straying onto them. The amount of waste residue to mix with the topsoil depends on the degradation rate, structure, and texture of the residue (Hillel, 1989). These factors, together with the amount of rainfall determine the time and space for retaining and degrading the contaminant. In an evaluation by Symons and Sims (1988), the extent and rate of detoxification of petroleum-contaminated soils undergoing landfarming treatment were directly related to waste loading rate. A previous study of the same soil in field conditions (Marin Milla´n, 2004) revealed that an initial application of 5–6% w/w total hydrocarbon content (TPH) on the topsoil showed the best result for degradation; thus, this was the leading rate applied in our landfarming experiment. Landfarming consisted of depositing the refinery sludge on the soil surface and mixing it with the top 1 m of the soil 1 week later by means of a tractor. Subsequent treatment simply consisted of aerating the top 1 m of soil once a month with a tractor. Control plots were treated in the same way as landfarming plots. Conditions were totally natural with no added water or nutrients; the minimum aeration was to ensure that the bioremediation experiment was as cheap as possible to run. At different times after adding the sludge (1,3,5,7,9,11 months) samples were taken from each of the plots. In the experimental plots, the sample was taken before each monthly aeration. Composite soil samples of the top 15 cm were randomly collected from six different places in each site. Each soil sample consisted of six 150-cm3 subsamples, which were thoroughly mixed to obtain a composite sample. The samples were brought to the laboratory on the same day and kept in the refrigerator at 4 C until they were analyzed (microbiological analysis were carried out 3–4 days after sampling). 2.3. Chemical parameters Electrical conductivity (EC) and pH were measured in a 1/5 solid/liquid aqueous extract. Total organic carbon was measured by the Bakley–Black method. In the oil sludge, chloride, nitrites, sulfate, ammonium, and fluor were measured by HPLC. TPH was measured by the infrared USEPA method (1983). Water-soluble carbon was measured in the extract 1/10 (solid/liquid) in automatic total organic
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carbon (TOC) analyzer. Water-soluble carbohydrates, in the same extract, were measured with anthrone by the Brink et al. (1960) method and soluble polyphenol compounds by the Kuwatsuka and Shindo (1973) method. 2.4. Microbiological analysis Microbial biomass carbon (MBC) was determined using a fumigation–extraction procedure (Vance et al., 1987). The 0.5 M K2 SO4 -extracted C was measured as indicated for water-soluble C. Basal respiration was determined in 50 g dry soil placed in hermetically sealed flasks, moistened to 50% of its water-holding capacity, and incubated in the dark at 28 C. The CO2 released was measured by a IR CO2 detector. The metabolic quotient ðqCO2 Þ was calculated by dividing the C–CO2 released from the sample in 1 h by the microbial biomass carbon content. The microbial biomass carbon to total organic carbon ratio (MBC/TOC) was also calculated. 2.5. Enzymatic assays Dehydrogenase activity. (DHA): Dehydrogenase activity was determined by the Skujins (1976) method as modified by Garcia et al. (1997). Soil ð1 gÞ at 60% of its field water-holding capacity was treated with 0:2 mL of 0.4% 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride in distilled water for 20 h at 22 C in darkness. The iodo-nitrotetrazolium formazan formed was extracted with 10 mL of a mixture of 1:1.5 ethylene/ chloride acetone by shaking vigorously for 1 min and filtering through a Whatman No. 5 filter paper. Iodonitrotetrazolium formazan was measured spectrophotometrically at 490 nm. Urease activity: Two milliliters of phosphate buffer (pH 7) and 0:5 mL of 6.4% urea were added to 0:5 g of soil and then the mixture was incubated at 30 C for 90 min before the volume was made up to 10 mL with distilled water. The ammonium released after addition of 0:1 mL 10 M NaOH was measured using an ammonium-selective electrode (CRISON micro pH 2002). A control without urea was used with each sample (Nannipieri et al., 1980). Protease activity on N-a benzoyl-L-argininamide (protease-BAA): Two milliliters of phosphate buffer (pH 7) and 0:5 mL of 0.03 M N-a-benzoyl-L-argininamide (BAA) substrate were added to 0:5 g of soil. The mixture was incubated at 37 C for 90 min and then diluted up to 10 mL with distilled water. The ammonium released was measured in the same way as that for urease (Nannipieri et al., 1980). Phosphatase activity: Two milliliters of 0.1 M maleate buffer (pH 6.5) and 0:5 mL of 0.115 M p-nitrophenyl phosphate were added to 0:5 g of soil and incubated at 37 C for 90 min. the reaction was stopped by cooling at
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2 C for 15 min and then 0:5 mL of 0.5 M CaCl2 and 2 ml of 0.5 M NaOH were added to the soil mixture before centrifugation at 4000 rpm for 5 min. The pnitrophenol formed was determined in a spectrophotometer at 398 nm (Tabatabai and Bremner, 1969). Controls were made in the same way, although the substrate was added before the CaCl2 and NaOH. b-Glucosidase activity: Two milliliters of 0.1 M maleate buffer (pH 6.5) and 0:5 mL of 50 M pnitrophenyl-b-D-glucopyranoside were added to 0:5 g of soil. Then the same procedure reported for phosphatase assay was follwed, except that the NaOH was substituted by Tris buffer, pH 12.
2.6. Ecotoxicity assay A toxicity test was carried out using luminescent bacteria (microtox), in which the inhibition of the luminescence of Photobacterium phosphoreum was measured using a luminometer (Kapanen and Ita¨vaara, 2001) after adding extracts of the samples. The microtox assay has been used to evaluate the rate and extent of detoxification of petroleum-contaminated soils under landfarming conditions (Symons and Sims, 1988). This assay uses a suspension of luminescent bacteria (P. phosphoreum) as bioassay organism for measuring acute toxicity in aqueous extracts of soil (Bulich, 1979; Matthews and Hastings, 1987). Lyophilized bacteria were used after rehydration in the commercial solution. All assays were carried out at 15 C with 15- and 30-min contact periods between 0:5 mL of bacterial suspension and soil suspension. Soil suspension was prepared by mixing 1 g soil with 10 mL of 2% NaCl (w/w) solution.
3. Results and discussion 3.1. Refinery sludge The main nonmetal compounds contained in the refinery sludge used in the experiment were organic compounds (hydrocarbons) (Table 1), especially alkanes, paraffins, aromatics, phenols, asphaltenes, and polynuclear aromatic hydrocarbons (Concawe, 1980). As is known, some of these compounds can be considered labile because they are easily degraded by microorganisms (simple aliphatic and aromatic compounds), while heavier components (such as asphaltenes) are more recalcitrant and difficult to degrade by bioremediation (Marin Milla´n, 2004). An important point is the high chloride content in the refinery sludge (Table 1), which is responsible for the high electrical conductivity values of the material, which means that great care should be taken if it is used for soil disposal as the excessive salt content may inhibit soil microbial activity (Garcia and Herna´ndez, 1996). Analysis of the heavy metal content of the semisolid refinery sludge indicated high quantities of Zn, Pb, and Ni. However, we must accept that the accumulation of such metals following the addition of refinery sludge does not necessarily have a negative effect on the microbial populations which carry out the hydrocarbon degradation processes, since studies on ecological doses in heavy-metal-contaminated soils have shown that very high levels of soluble metals are needed to negatively influence the soil’s metabolic activity (Moreno et al., 2003). More problematic in this sense can be the quantity of phenols found in the sludge, since they may well have a negative effect on the microbial population responsible for hydrocarbon degradation (Concawe, 1980), or even salinity, which may, as we have already pointed, alter such microbial activity.
2.7. Statistical analysis 3.2. pH and electrical conductivity in landforming soils Statistical analysis (ANOVA analysis and LSD test for mean) was performed with the Statgraph program on data obtained in the control and landfarming plots.
The bioremediation of hydrocarbons of the refinery sludge will be carried out by soil microorganisms; so soil
Table 3 pH and electrical conductivity (EC) in the landfarming and control plots Time elapsed after oil sludge addition (months)
EC ðdS m1 Þ
pH Control plots
Landfarming
Control plots
Landfarming
1 3 5 7 9 11
7.70 8.24 8.68 8.07 8.62 8.68
7.59 7.57 7.50 7.56 7.61 7.65
0.90 0.49 0.60 1.09 0.40 0.27
3.38 3.48 2.12 2.20 2.79 2.71
LSD ðPo0:05Þ
0:51
0:32
0:35
0:45
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characteristics that influence soil microbial activity such as pH will also influence hydrocarbon degradation. Most heterotrophic bacteria and fungi need a pH near neutrality, with fungi being more tolerant of acidic conditions (Atlas, 1988). Extreme pH values could have a negative effect on the ability of microbial populations to degrade hydrocarbons. Dibble and Bartha (1979) observed an optimal pH of 7.8 for the mineralization of oily sludge in soil. In our case, the pH values of the landfarming plot ranged between 7.5 and 7.65, a range suitable for microbial growth (Table 3). Soils with oil sewage showed higher EC values than control soils (Table 3). There are few published studies dealing with the effects of salinity on the microbial degradation of hydrocarbons. Shiaris (1989) reported a general positive correlation between the salinity and the rates of mineralization of phenanthrene of naphtalene. Kerr and Capone (1988) observed a relationship between the mineralization rate and the salinity in sediments of the Hudson river. In contrast, Ward and Brock (1978) showed that rates of hydrocarbon metabolism decreased with increasing salinity in the range 3.3–28.4% and attributed the results to a general reduction in microbial metabolic rates. In our case, and according to previous studies (Garcia and Herna´ndez, 1996), the salinity detected in the landfarming plots (Table 3) could have a negative effect on the microbial activity in these soils and so on hydrocarbon bioremediation, especially when the salinity is due to chlorides as opposed to sulfates.
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3.3. Carbon fractions in landfarming soils Fig. 1 shows that the content of total organic carbon at the beginning of the experiment was greater in the landfarming than in the control plots, although these levels decreased by 27% during the experiment. It was to be expected that carbon compounds would be degraded to a greater extent during the first months of landfarming because of the aeration carried out which should favor the mineralization of the organic matter and provoke the degradation of the hydrocarbons contained in the refinery sludge. It is possible that the technique used to measure the TOC (Wakley–Black method) produces an error, especially at the outset, when there are more labile hydrocarbons that may be affected by the temperature reached with this method when soil samples are treated with sulfuric acid. The most labile fractions of C (polyphenolic compounds and water-soluble carbohydrates and carbon) constitute some of the most immediate energy sources of microorganisms and their levels reflect microbial activity in general (Garcia et al., 2000). These fractions are continually formed and degraded due to the mineralization of the organic matter and microbial synthesis. The addition of refinery sludge increased the proportion of these labile C fractions compared with that found in the control soil (Fig. 1). The behavior of these carbonaceous fractions in the landfarming plots was similar, the high values at the outset diminishing since they were constantly used as energy source by the microorganisms. The successive aeration would have contributed to the
Fig. 1. Content of different carbon fractions in the landfarming and control plots.
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mineralization of the carbon compounds, as has been confirmed by several authors in studies of turning agricultural soils (Haynes and Tregurtha, 1999; Hussain et al., 1999). In the last stages of landfarming these fractions tended to be partially regenerated, perhaps because of the decomposition of other, more recalcitrant carbon molecules. This could explain why, at the end of the experiment, the values obtained for the labile carbon fractions exceeded those of the control soils. In the case of the polyphenols, their disappearance rate was slower than those of the water-soluble carbon and watersoluble carbohydrates and it took 7 months to reach their minimum level since their more complex structure makes them more resistant to biodegradation. This is not a positive finding since some studies (Concawe, 1980) mention that a high concentration of phenols in a soil has a negative effect on the microbiol populations capable of degrading hydrocarbons.
3.4. Hydrocarbons content in landfarming soils Fig. 2 shows the soil hydrocarbon content as a function of time. It is clear that the bioremediation method followed (simple aeration of the soil with the sludge) significantly reduces the hydrocarbon content of the sludge (80% reduction in 11 months). Aliphatic and aromatic hydrocarbons are degraded in the highest quantity according to Marin Milla´n (2004). The initial steps in the catabolism of aliphatic, cycling, and aromatic hydrocarbons by bacteria and fungi involve the oxidation of the substrate by oxygenases, for which molecular oxygen is required. Aerobic conditions are therefore necessary for this route of microbial oxidation of hydrocarbons in the environment. Under semiarid conditions with scarce rain, the biodegradation of hydrocarbons has taken place in 11 months. This time could probably be shorter if soil humidity (in our case,
Fig. 2. Hydrocarbons content of the soil during landfarming experiment (LSD, Po0:05 : 0:12 for landfarming plots and 0.08 for control soil).
1–3%) is higher, but it is not possible under semiarid conditions. The loss of organic compounds in landfarming plots is governed by a first-order kinetics, the elimination rate of a compound being proportional to its concentration (Demque et al., 1997). During the degradation process two clearly differentiated phases were observed: a first stage with a high velocity, in which the hydrocarbon degradation rate was maximal (55% of the total hydrocarbon degradation takes place in 2 months, from month 1 to month 3) and the most labile fractions (mainly aliphatic) were involved, and a second slower stage (45% of the total hydrocarbon degradation takes place in 8 months, from month 3 to month 11). The first stage lasted 2 months, after which the degradation rate slowed down, demonstrating that, as the most easily biodegradable hydrocarbons are consumed, the microorganisms turn their attention to other fractions, such as the aromatic, condensed cyloalkanes, etc., which are degraded at different rates (Langbehn and Steinhart, 1995). After 9 months the degradation rate slowed down sharply, since the remaining fractions are structurally more complex hydrocarbons and therefore less accessible, their recalcitrance and low bioavailability causing the activity of the microbial populations to drop (Margesin et al., 1999). Sims and Sims(1999) also mentioned the biphasic kinetics of petroleum hydrocarbons during degradation by landfarming. This degradation rate depends on, among other factors, the ability to control environmental variables. For example, temperature influences petroleum biodegradation by its effect on the physical nature and chemical composition of the oil, rate of hydrocarbon metabolism by microorganisms, and the composition of the microbial community (Atlas, 1981). 3.5. Metabolic activity in landfarming soils In a soil where a landfarming process is being carried out (for example, for reducing its hydrocarbon content as in our case), the microbial population and its activity could be influenced for that process (quantity of toxic in the soil); therefore, the study of some parameters indicative of microbial activity can be of interest. The microbial biomass carbon includes only 0.5–4.6% of the soil TOC content but is responsible for the decomposition of elements involved in soil fertility (Dı´ az-Ravin˜a et al., 1993). MBC has a turnover rate that is several times faster than that of organic matter and so the flow of nutrients from this fraction is greater than that from others, which explains its influence on the availability of nutrients to plants (Sparling, 1992). In our experiment the landfarming plots showed MBC values that were considerably above those of the control soils (Fig. 3), demonstrating that microbial biomass size and activity increased with the incorporation of the sludge. At the
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seemed to be closely correlated with that of MBC during the landfarming process. The ATP content was also higher at all times in the soil with refinery sludge than in the control soil, especially during the first months, when the rate of metabolic activity was at its highest. During this time the microorganisms that are responsible for degrading the hydrocarbons set to work and increase in numbers. These microbial populations diminish in size as the substrates are used up and mineralized, the ATP content decreasing since it can exist only inside living cells. Soil respiration measurements give an idea of the microbial activity in the soil and of the quantity and quality of substrates susceptible to mineralization (Anderson, 1982). In our experiment, the microbial activity was high just after addition of the refinery sludge since the most labile hydrocarbon compounds present need high microbial activity if they are to be degraded (Fig. 3); hence, the increase in respiration recorded. CO2 emission gradually decreases as the most labile hydrocarbon fractions disappear, with only the most recalcitrant fractions remaining. However, such behavior is very susceptible to humidity and temperature (Leiros et al., 1999). Humidity, for example, conditions the capacity of microorganisms to mineralize organic matter, which is why long periods of drought like those occurring in the study area decrease soil microbial activity (Orchard and Cook, 1983). Temperature has both a direct and an indirect effect on such processes. (Davidson et al., 1998). In our case, the minimum temperature were never low enough to stop respiration, although the maximum temperatures, for their indirect effect on drought, could be said to have a negative influence. All parameters studied indicate that microbial activity diminishes with the bioremediation process but is always higher than that of the control soil. 3.6. Metabolic quotient and MBC/TOC ratio100 Fig. 3. Microbiological parameters in the landfarming and control plots.
beginning of the experiment some sort of inhibition that affects MBC seems to exist; this is not unexpected given the large quantity of exogeneous organic matter and high amount of hydrocarbon added. It is known that this parameter is subject to seasonal variations (Nannipieri et al., 1990), although we think that any variation in this sense is much less than the influence of the addition of the refinery sludge. Nannipieri et al. (2002) also indicated that climates with wide thermal variations (such as dry tropical) seemed to have the greatest influence on seasonal variations of microbial biomass. Another parameter that can be used to measure the overall microbial activity of a soil is the adenosin-5triphosphate (ATP) content (Fig. 3), whose behavior
The ðqCO2 Þ reflects the efficiency with which the carbon is used. This parameter can discriminate between the maturity levels of ecosystems since it is assumed that mature systems respire less per unit of biomass because they canalize less energy toward their metabolism. It can also act as an index of stress in the face of contamination (Wardle and Ghani, 1995; Insam and Domsch, 1988; Anderson and Domsch, 1993). The values of qCO2 increased very significantly in the landfarming plots after the addition of the sludge (Table 4), which agrees with the observations of other authors such as Marin Millan (2004), who suggested that a disturbance (as the addition of sludge in our experiment might be interpreted) can increase the metabolic coefficient. The reason for this in our case would be that microorganisms must raise their respiration per unit of biomass to
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Table 4 Metabolic quotient (qCO2), MBC=TOC 100 ratio, and dehydrogenase activity (DHA) in the landfarming and control plots Time elapsed after sludge oil addition (months)
1 3 5 7 9 11 MDS Po0:05
qCO2(mg C–CO2mg MBC g1 h1 )
MBC=TOC 100
Control
Landfarming
Control
Landfarming
Control
Landfarming
6.6 4.1 2.3 5.1 2.8 4.2 2:3
1.36 8.7 6.6 9.9 8.3 11.9 2:9
0.56 1.08 0.44 0.23 0.84 1.43 0:34
7.29 5.15 2.42 1.46 1.29 1.77 0:45
73.0 32.0 70.7 35.4 43.1 38.9 10:3
197.1 156.8 99.4 90.6 89.0 62.6 21:5
degrade the hydrocarbons. At the end of the experiment, the qCO2 values did not fall, perhaps because the system was not reaching a certain degreee of equilibrium after the disappearance of the easily biodegradable hydrocarbons during the first months. The MBC/TOC ratio was higher in the soils receiving the sludge than in the control soils (Table 4). It is clear that the microbial biomass increases in quantity to help in the biodegradation of substrates such as hydrocarbons. At the end of the landfarming process, when most of the easily biodegradable hydrocarbons present in the refinery sludge have been degraded by the microorganisms existing in the soil, the ratio decreased. The existence of a minor quantity of available substrates for microorganisms could be responsible for this result. 3.7. Enzyme activities Soil enzymes are biological catalysts of specific reactions and these reactions, in turn, depend on a variety of factors. Numerous studies of enzymes exist and comparisons have been made in various soils subjected to different climatic conditions and management practices (Burns, 1978; Ladd, 1985). Nonpolar organic compounds, such as hydrocarbons, are hydrophobic and do not interact significantly with proteins in solution. In soil, crude oil at high concentrations ð100 kg m2 Þ may block the expression of enzyme activity by coating organomineral and cell surfaces and thereby prevent soluble substrates from reaching the enzyme molecules (Speir and Ross, 2002). These authors indicate that, at moderate levels of hydrocarbon contamination, some enzyme activities declined and some increased. Dehydrogenase activity has been used to assess microbial activity, although this use has been criticized by different authors; Benefield et al. (1977) indicated that DHA is not an accurate parameter for determining the electron flow rate to O2 because the electron acceptors used in the dehydrogenase assays are less efficient than O2. However, Garcia et al. (1997) found that DHA is a good index of the status of soil microbial
DHA (ng INTF g1 )
activity in semiarid Mediterranean areas. Our data confirm that DHA is higher in landfarming plots than control soil probably due to higher microbial activity in these plots. DHA has been widely used to measure the catabolic activities in soil, which are correlated with microbial activity (Skujins, 1976). In the landfarming plots, DHA diminishes with the time; when hydrocarbons have already been degraded, microbial activity decreases and DHA is lower. The hydrolases belonging to the cycles of elements such as nitrogen (urease and protease activity which hydrolyse BAA), phosphorus (phosphatase activity), or carbon (b-glucosidase activity) were determined in the control soil and in those with the refinery sludge subjected to landfarming (Fig. 4). In the first 3–5 months the activity in the landfarming soils was higher than that in the corresponding controls. As Rastin et al. (1998) pointed out, enzymatic activities are influenced by the biological matter of the soil, in our case by the addition of the refinery sludge and the changes in the soil as a result of landfarming. It is clear that the cycles of the most important elements in the soil are affected by the introduction of the refinery sludges. After the most labile hydrocarbons of the sludge have been degraded, the enzymatic activities return to values similar to those of the control soil (Fig. 4).
3.8. Ecotoxicity with luminiscent bacteria This toxicity assay provides information complementary to the microbial activity measurements described above. In fact, bioassays are generally used to check for toxic elements that are bioavailable for exogenous organisms (Ro¨nnpagel et al., 1998). Concawe (1980) summarized the environmental toxicological effects of petroleum refinery effluents and found that, in general, oils increase in toxicity with increasing levels of lowboiling compounds, unsaturated compounds, and aromatics. Also aromatics with increased numbers of alkyl substituents have higher toxicity, and toxicity increases along the series alkanes–alkenes–aromatics.
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Fig. 4. Enzyme activities in the landfarming and control plots.
Fig. 5. Percentage of inhibition on luminescent bacteria during landfarming experiment (LSD, Po0:05 : 14:5 for landfarming plots and 2:1 for control soil).
Cycloalkanes and cycloalkenes appear to be more toxic than alkanes. Fig. 5 represents the percentage of the inhibition of luminescent bacteria. As can be appreciated, the level of inhibition decreased dramatically in the soils subjected to landfarming. As the extracts treated with this method are aqueous extracts, these findings indicate that the water-soluble toxic compounds are totally sanitized after 9 months.
conditions. The biodegradation of total hydrocarbons has been established at 80% in 11 months. Dry conditions in semiarid climate, where the rain is very scarce, can be responsible for this behavior. In general, parameters indicative of soil microbial activity measured during the process of hydrocarbon degradation showed higher values than the control soil; nonpolar organic compounds (as hydrocarbon content in refinery sludge) are hydrophobic and do not interact significantly with proteins in solution, and for this reason enzyme activities are not particularly inhibited under these conditions. After the most labile hydrocarbons of the sludge have been degraded, the enzymatic activities diminish.
Acknowledgements The research was financially supported by RepsolYPF (Research Contract between Repsol-YPF and CEBAS-CSIC). The authors thank Mr. Jesus Mellado, Mr. Francisco Navarro, and Mr. Angel Crespo for their help with the manuscript.
References 4. Conclusion In conclusion, we can stay that the bioremediation of refinery sludge by landfarming is possible is semiarid
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