evaluation of optimum concentration of two anionic surfactants in the ...
Ministério da Ciência e Tecnologia Coordenação de Processos Metalúrgicos e Ambientais
EVALUATION OF OPTIMUM CONCENTRATION OF TWO ANIONIC SURFACTANTS IN THE BIODEGRADATION OF CRUDE OIL CONTAMINATED SOIL
Valéria Souza Millioli Letícia Cotia dos Santos Andréa C. de Lima Rizzo Ronaldo Luiz C. dos Santos CETEM
Adriana U. Soriano CENPES/PETROBRAS
CT2005-075-00 – Comunicação Técnica ao ConSoil 2005, 3 -7 de outubro de 2005, Bordeaux, França Outubro / 2005
ConSoil 2005 EVALUATION OF OPTIMUM CONCENTRATION OF TWO ANIONIC SURFACTANTS IN THE BIODEGRADATION OF CRUDE OIL CONTAMINATED SOIL Millioli, Valéria Souzaa,b,; Santos, Leticia Cotia dosa,b; Rizzo, Andrea Camardella de Limaa,b; Soriano, Adriana UC.; Santos, Ronaldo Luiz Correa dosa. a)Center for Mineral Technology/ Ministry of Science & Technology (CETEM/MCT)
b) School of Chemistry/ Federal University of Rio de Janeiro (EQ/UFRJ) c) Petrobras Research Center (CENPES) Mail address: Avenida Ipê, no 900 - Ilha da cidade Universitária,Rio de Janeiro – RJ- Brasil - CEP: 21941-590; E-mail:[email protected]; Phone+ 55 (21) 3865-7282; Fax: + 55 (21) 3865 7232; PhD student
Abstract Hydrophobic Organic Compounds (HOCs) present in crude oil are quite insoluble in aqueous phase and hence are not available towards hydrophilic microorganisms. Addition of surfactants to enhance effectiveness bioremediation on contaminated soil has been investigated in order to increase the bioavalaibility of several organic contaminants. In Bioremediation, the use of slurry bioreactor has received more attention because it requires a relatively small space and exhibit faster rates of biodegradation when compared with others biotechnological systems. The aim of this research was to improve the bioremediation of a contaminated soil in through the addition of surfactants. For this purpose, two anionic surfactants were investigated: a chemical and a biological one (rhamnolipids). Concentrations of those surfactants were based on their active ingredient and varied on 0.15% up 1.5% w/w. Assays were conduced weighting 300g of dried soil in a cubic flasks with 600 cm3 capacity. Soil samples were mixed with water (50% of field capacity), nutrient enrichment and surfactants on varied concentrations. Experiments were monitored for 60 days, in terms of Oil and Grease (O&G) removal. It was verified that the biodegradation was more effective, after 60 days, with adding biological surfactant (30% of O&G removal) in the concentration of 0.6 % of active ingredient (rhamnolipids). Addition of chemical surfactant as an auxiliary to the bioremediation process increased biodegradation in terms of O&G removal for the concentrations between 0.3% up 1.5%, but no further was observed as a consequence of addition of 0.15% w/w of active ingredient, in relation to the bioestimulated control (5% of O&G removal). After 30 days it was verified a decrease of O&G removal for all experiments. Within the first 30 days the best result was obtained with 0.3% of chemical surfactant (23% of O&G removal) while to the 60 days, this same condition presented 9.5% of O&G removal. Key –words: Biosurfactant, chemical surfactant, petroleum, bioremediation, soil 1. INTRODUCTION Oil pollution from industrial sources, shipping operations and others activities cause a great environmental impact to terrestrial and marine ecosystems. The contamination of soil by hazardous organic pollutants is a current environmental problem. Various approaches have been proposed for treating petroleum hydrocarbon contaminated soil. Among these treatments, the bioremediation technologies have been shown to be a promising technology wherein microorganisms are stimulated to metabolize the crude oil adsorbed in soil matrix (Nano et al. 2003; Sabaté et al. 2004 ). The biological treatment of an oil-contaminated soil can be affected by hydrocarbons structure and weathering processes, which decrease the bioavailability of pollutants to microorganisms. Weathering refers to the result of biological, chemical and physical processes that can affect the type of hydrocarbons that remain in soil (Troquel et al. 2003). Those processes also enhance the sorption of hydrophobic organic contaminants to the soil matrix, decreasing the rate and extent of biodegradation (Bosma et al. 1997). Moreover, a weathered oil-contaminated soil normally contains a recalcitrant
fraction of compounds composed, basically, of high molecular weight hydrocarbons (higher than C25 compounds), which could not be easily degraded by indigenous microorganisms. In contrast, a recently oil-contaminated soil contains a higher amount of saturated and aliphatic compounds, which are the most susceptible to the microbial degradation. Many studies have been demonstrated that the additions of surfactant in the bioremediation process may enhance the oil mobility and increase its availability, improving the biodegradation rates (Alexander, 1994; Kim et al. 2001; Rahman et al. 2003). Surfactants are surface-active agents composed of amphipathic molecules consisting of hydrophilic polar head moiety and a hydrophobic nonpolar tail moiety. They reduce the surface tension by forming an extremely small aggregate that is called micelles. At low concentrations, surfactants are soluble in water, and when increasing their concentration, the molecules form micelles in solution. The smaller concentration at which micelles begin to form is called as the Critical Micelle Concentration (CMC). Above the CMC value, the surfactants have been reported to solubilize petroleum hydrocarbons in soil-water systems, increasing the biodegradation rates (Aronstein & Alexander, 1992; Alexander, 1994; Deshphande, 1999). 2. OBJECTIVE The aim of this work was evaluate the influence of addition of two different anionic surfactant in the treatment of petroleum contaminated soil. 3. MATERIALS AND METHODS 3.1. SOIL SAMPLE Contaminated soil was extracted two years after an oil spill accident occurred in São Paulo (Brazil).. Soil was homogenized, then a representative sample was taken, and further stored at ± 4° C to avoid natural degradation. The chemical and physical properties of the soil are given in Table 1.
Table 1. Chemical and physical properties of contaminated soil Parameter
Soil content
Parameter
Soil content
C organic
46g/kg
pH
5.1
N
1.0 g/Kg
Plasticity
38 %
P
0.001g/Kg
TPH
22 mg/g
K
0.084g/Kg
HPA’s
4.4mg/g
Organic matter
13.7 %
O&G
3.3 %
Sand
59 %
Field Cap. - F/C
38. 5 %
Clay
24 %
Silt
19 %
3.2. SURFACTANTS Two different anionic surfactants were chosen to be evaluated for these experiments: a chemical and a biological (rhamnolipids) one. Concentration range of these surfactants applied to the contaminated soil was based on active ingredient corresponding: 0.15 up 1.5 % w/w (in relation to soil mass) as shown in Table 2.
3.3. EXPERIMENTAL SET-UP Into a glass cubic flask of 600 cm3 capacity, were added 300g of dried soil. Soil samples were neutralized with Ca(OH)2 and then supplemented with nutrients based on optimum nutritional ratio C:N: P = 100:2,5:1. This ratio, had been established in others essays as excellent for this soil biodegradation (Trindade et al, 2005). Enrichment with nutrients was made through the addition of KH2PO4. Humidity was adjusted to 50% of soil Field Capacity. Assays were incubated at 30°C, during 60 days and periodically checked for pH analysis, aeration. When shown necessary, new correction of nutrients, pH and humidity were done. Surfactants for each experiment were added as shown in Table 2. Table 2: Conditions used in bioassays with the addition of surfactants (that contained 300g of soil, correction of nutrients (C:P = 100:1), pH = 7 and humidity (50% of soil the field capacity)): Conditions
Concentration of chemical surfactant
Concentration of biological surfactant
Control
-
-
1
-
0.15% p/p
2
-
0.3% p/p
3
-
0.6% p/p
4
-
0.9 p/p
5
-
1.5% p/p
6
0.15% p/p
-
7
0.3% p/p
-
8
0.6% p/p
-
9
0.9 p/p
-
10
1.5% p/p
-
3.4. ANALYTICAL METHODOLOGIES 3.4.1. Apparent Critical Micelle Concentration (ACMC) and Critical Micelle concentration (CMC) Measurements on surface tension were carried out accordingly in different dilutions of surfactants in a Soil-Water-Oil (SWO) systems. Surfactant solutions had been mixed in a ratio 1:10 (soil:solution). These mixtures were agitated in a shaker during 16 h. After agitation, mixtures were centrifuged at 3000 rpm during 10 minutes. Supernatants were filtered to verify only the solubilized oil after surfactant applications. Surface tension was measured using ring method in a digital-Tensiometer K10T (Kruss, Hamburg, Germany). The ACMC value expressed in mg/L was obtained from the plot of surface tension versus surfactant concentrations solution. 3.4.2. pH Determination 20g of soil was removed from each experiment, in interval of 30 days, and mixed with 50mL of distilled water. After that, the samples were agitated in a shaker during 1h and then the pH value was measured.
3.4.3. Oil and grease (O&G) determination Oil and grease determination was quantified by gravimetric method, extracting 2g of dried soil with hexane by ultrasound (Rizzo & Raimundo, 2003). 3.4.4. Quantification of microbial population Quantification of the final and initial microbial population was made by the pour plate technique. The results were expressed in terms of Counting Forming Unit (CFU)/g of soil (Uruhay, 1998). 4. RESULTS AND DISCUSSION 4.1. EVALUATION OF THE BIODEGRADATION BY O&G REMOVAL Figure 1 shows the results of oil and grease (O&G) removal in the bioassays when it had been added a biological surfactant as well as the results of biodegradation of the biostimulated and the sodium azide control (abiotic control). It was verified that, after 60 days, the highest O&G removal was obtained when 0.6 % of rhamnolipid was applied (30% of O&G removal). Except to the experiment using 1.5% (w/w) of rhamnolipids, all others condition tested had shown a tendency to reach greater oil removal above 60 days. In those assays the results of biodegradation varied of 19.2 up to 30%, being results very above of the biostimulated (5%) and the azide (0.8%) control ones, proving the effectiveness of the biosurfactant addition. 35
0.15% 0.3% 0.6% 0.9% 1.5% Control azide control
O&G removal (%)
30 25 20 15 10 5 0 0
10
20
30
40
50
60
70
Time (days)
Figure 1: Evaluation of O&G removal (%) after the addition of biosurfactant in different concentrations On the same way, Figure 2 shows results on O&G removal in the bioassays in which it was added a chemical surfactant. It was observed, after 30 days, that there was a decrease of oil and grease removal for all experiments. Until the first 30 days the best result was obtained when 0.3% of chemical surfactant was applied (23% of O&G removal). However, at the end of the experiment (after 60 days)it dropped until a level of 9.5% of O&G removal.
0.15% 0.3% 0.6% 0.9% 1.5% control azide control
25
O&G removal (%)
20
15
10
5
0 0
10
20
30
40
50
Time (days)
60
70
Figure 2: Evaluation of O&G removal (%) after the addition of chemical surfactant in different concentrations Results presented in Figure 3 (A and B) confirm which were the best conditions with the addition of surfactants (biological and chemical one), after 30 and 60 days. It was observed on Figure 3A that in first 30 days of biodegradation, the higher was the biosurfactant concentration, the highest was the oil and grease removal. However, after the 60 days, it was an optimum concentration, as already previously, said (0.6% of rhamnolipid concentration). It was still observed, after 60 days, experiments, using 0.9 % and 1.5% had a decrease of O&G removal in relation to the best condition (0,6% of rhamnolipid). A decrease of O&G removal of these experiments could be associated to an extreme agglomeration of the biosurfactant with the soil matrix. Maybe, this extreme agglomeration of the biosurfactant could contribute for reduction of the oxygenation for the aerobic microorganisms, decreasing the effectiveness of the biodegradation process. Another factor should be considered is that high biosurfactant concentrations could be toxic to the biodegradation system. The Figure 3B shows that the best condition was the 0.3% after 30 days, as previously said. 35
25
23
30
30
30 days 60 days
25,5
25 20 15 30 days biological 60 days biological
10
O&G removal (%)
O%G Removal (%)
20
15 9,5
10
5
5 0 0
0,2
0,4
0,6
0,8
1
surfactant (%)
A
1,2
1,4
1,6
0 0
0,5
1
1,5
chemical surfactant concentration (%p/p)
B
Figure 3: The best condition on 30 and 60 days after the addition of the biosurfatant (A) and the chemical surfactant (B).
2
4.2 – EVALUATION OF CMC AND ACMC IN RELATION TO THE OIL AND GREASE REMOVAL Analyzing the two surfactants employed, the chemical surfactant had an ACMC value greater than the biological one, as can be seen in Table 3. These characteristics of biosurfactants to have low ACMA and /or CMC values have being investigated for some authors (Stelmack et al., 1999; Banat, 1995). The CMC value for these different surfactants can varies so much with their structure, but others factors like the temperature of the solution and the presence of organic compounds can affect the CMC value (Haigh, 1996). Surfactants that have low CMC and ACMC value are more appreciable because they can be used in a fewer amounts in relation to others with high CMC and ACMC values, to get similar effect. ACMC value was much higher than the CMC value for both surfactants investigated in the soil-water-oil (SWO) system (Table 3). In the SWO systems the CMC value is modified because there are an extensive adsorption of surfactants onto soil surfaces, which means that much more surfactants are required in soil that might be expected to reach the CMC in systems utilizing clear water (Haigh, 1996). The ACMC values of chemical surfactant were above the biological one. ACMC and CMC values are important, since the addition of surfactant was evaluated in relation to the active ingredient and not on the ACMC value. The ACMC values of chemical surfactant were above the biological one. So, the chemical surfactant should be added in these bioassays in a bigger concentration than the biological one (about 5 times, in accordance with the ACMC values- Table 3), increasing most probably operational cost. Table 3. CMC and ACMC values for each surfactant Surfactant
CMC (mg/L)
ACMC (g/L)
Chemical
100
5
Biological
43
1
4.3 – EVALUATION OF MICROBIAL POPULATION IN RELATION TO THE OIL AND GREASE REMOVAL It were observed in Table 4 that the sodium azide control (abiotic control) did not present microorganisms population in the soil, along the 60 days, as was expected. Biostimulated control kept the same order of microorganisms population magnitude along the 60 days. Experiment with 0.3% w/w of chemical surfactant and 1.5% w/w of rhamnolipids decrease in microorganism population after 60 days (in relation to the 30 days) and this had also reflected in the efficiency of O&G removal. These results are in accordance with those previously presented in Figure 1, which indicated a decrease in O&G removal after 60 days of biodegradation process. In the experiment with 0.6% of rhamnolipid occurred an increase in microorganisms population after 30 days remaining the same magnitude after 60 days, indicating that these microorganisms could biodegrade more oil above the 60 days, as shown in Figure 1. Table4: Microorganisms population of the best condition MICROORGANISMS (UFC/g SOIL) t=0
t=30 days
T = 60 days
Azide control
0
0
0
Control
1.4x106
4.6 x106
3.4x106
0.3% (chemical)
3.2x106
2.5x106
4.5x105
0.6% (biological)
1.6x106
6.1x108
3.0x108
1.5%(biological)
2.8x106
2.1x107
2.5x106
CONDITIONS
4.6 – EVALUATION OF pH ALONG THE 60 DAYS OF BIODEGRADATION During the biodegradation process all the experiments had been monitored by pH value and humidity, so as to keep humidity in 50% of the capacity of field and pH near 7.0. Figure 5 shows profiles for pH with addition of chemical surfactant (A) and biosurfactant (B). It can be observed in Figure 4A and 4B that it was not necessary to adjust the pH along the 60 days, since the pH values were kept between 6 and 7.4 during the biodegradation process.
A
8 7 6 5
0 day 30 days 60 days
pH
4 3 2 1 0 0.15
0.3
0.6
0.9
1.5
% chemical surfactant
9
B
8 7
0 days
6
pH 5
30 days
4 3
60 days
2 1 0
0.15
0.3
0.6
0.9
1.5
Biosurfactant concentration (p/p)
Figure 5. Soil samples pH with additions of chemical surfactant (A) and biosurfactant (B) 5 . CONCLUSIONS Additions of a biosurfactant as an auxiliary to the bioremediation process increased the biodegradation in terms of oil and grease removal for all concentrations added. This improvement can be seen in relation to the biostimulated control (without addition of biosurfactant), whose biodegradation rates was 5%. Biodegradation measurement for all experiments which have addition of biosurfactant varied between 19.2 up 30 % of O&G removal after 60 days. Addition of a chemical surfactant as an auxiliary to bioremediation process increased the biodegradation in terms of oil and grease removal when using concentration between 0.3% up 1.5% w/w, but no further was observed in addition of 0.15% w/w of active ingredient, in relation to the bioestimulated control. After 30 days it was verified a decrease of oil and grease removal for all
experiments. After 30 days the best condition was 0.3% of chemical surfactant (23% of O&G removal) and after 60 days, this same condition presented 9.5% of O&G removal. After 60 days of biodegradation process the best condition was the addition of 0.6% of rhamnolipids in the soil . This experiment presented tendency to increase the biodegradation rates above 60 days , as well as the tendency in increasing the microorganism population. Determinations of ACMC and CMC values were necessary to know the surfactants characteristics and the best conditions to use them. Great additions of surfactant are necessary when surfactants have high CMC and/or ACMC values, in comparison with other surfactants which have lower values. 6- ACKNOWLEDGEMENTS This work was supported by Center of Mineral Technology (CETEM) in partnership with CENPES – Petrobras. The authors wish to thank Ms. Marisa Monte and Antonieta Middea (CTM/CETEM) for the CMC and ACMC tension analysis and Luiz Sobral for the very rich discussing regarding this paper. 7- REFERENCES Alexander, M. Biodegradation and Bioremediation. Academic Press. California. 302p. (1994). Aronstein, B.N. and Alexander, M. “Surfactants at low concentrations stimulate biodegradation of sorbed hydrocarbons in samples of aquifer sand and soil slurries”, Environ. Toxicol. Chem. , 11, 1227-33. (1992). Banat, I.M. ‘Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: A review”. Bioresource Technology, 51, 1-12. (1995). Bosma TNP, Middeldorp PJM, Schraa G & Zehnder AJB. Mass transfer limitation of biotransformation: quantifying bioavailability. Environmental Science & Technology, 31(1): 248252. (1997). Deshpande, S. Shiau, B.J.; Wade, D.; Sabatini, D.A. and Harwell, J.H. “Surfactants selection for enhancing ex situ soil washing”. Pergamon., 33 (2), 351-60. (1999). Haigh, S.D. “A review of the interaction of surfactants with organic contaminants in soil”. The Science of the Total Environment, 185, 161-70. (1996). Nano, G.;Borroni, A. Rota, R. Combined slurry and solid-phase bioremediation of diesel
contaminated soils . Journal of Hazardous Materials, 100, 79-94. 2003. Rahman, K.S.M., Rahman, T.J.; Kourkoutas, Y. Petsas, I. Marchant, R. Banat, I.M. Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipids and micronutrients. Bioresourse Technology, 90, 159-168. 2003
Rizzo, A.C.L; Raimundo,R. Determinação de óleos e graxas, em solo, por gravimetria empregando método de extração com ultrassom”. IT2003-001-00 – Instrução de Trabalho elaborada para o CETEM. Sabaté, J. Viñas, M. and Solanas, A. M. Laboratory-scale bioremediation experiments on hydrocarbon-contaminated soils. International Biodeterioration & Biodegradation, 54, 19-25. 2004.
Stelmack, P.L.; Gray, M.R. and Pickard, M.A. “Bacterial Adhesion to soil contaminants in the presence of surfactants”. Applied and Environmental Microbiology, 65, (1), 163-8, Jan. (1999).
Trindade, P. V. O., Rizzo, A. C. L., Soriano, A. U., Sobral, L. G. S. and Leite, S. G. F. Bioremediation of a weathered and a recently oil-contaminated soi ls from Brazil: a comparison study. Chemosphere, V. 58, Issue 4 , January 2005, Pages 515-522. Troquel J, Larroche C & Dussap CG. Evidence for the occurance of an oxygen limitation during soil bioremediation by solid-state fermentation. Biochemical Engineering Journal, 13:103-112.(2003) Ururahy, A. F. P. Biodegradação de Resíduo Oleoso Proveniente de Refinaria de Petróleo. Tese DSc., Universidade Federal do Rio de Janeiro, Escola de Química, Rio de Janeiro, Brasil, 344p. (1998).
EVALUATION OF OPTIMUM CONCENTRATION OF TWO ANIONIC SURFACTANTS IN THE BIODEGRADATION OF CRUDE OIL CONTAMINATED SOIL
Valéria Souza Millioli Letícia Cotia dos Santos Andréa C. de Lima Rizzo Ronaldo Luiz C. dos Santos CETEM
Adriana U. Soriano CENPES/PETROBRAS
CT2005-075-00 – Comunicação Técnica ao ConSoil 2005, 3 -7 de outubro de 2005, Bordeaux, França Outubro / 2005
ConSoil 2005 EVALUATION OF OPTIMUM CONCENTRATION OF TWO ANIONIC SURFACTANTS IN THE BIODEGRADATION OF CRUDE OIL CONTAMINATED SOIL Millioli, Valéria Souzaa,b,; Santos, Leticia Cotia dosa,b; Rizzo, Andrea Camardella de Limaa,b; Soriano, Adriana UC.; Santos, Ronaldo Luiz Correa dosa. a)Center for Mineral Technology/ Ministry of Science & Technology (CETEM/MCT)
b) School of Chemistry/ Federal University of Rio de Janeiro (EQ/UFRJ) c) Petrobras Research Center (CENPES) Mail address: Avenida Ipê, no 900 - Ilha da cidade Universitária,Rio de Janeiro – RJ- Brasil - CEP: 21941-590; E-mail:[email protected]; Phone+ 55 (21) 3865-7282; Fax: + 55 (21) 3865 7232; PhD student
Abstract Hydrophobic Organic Compounds (HOCs) present in crude oil are quite insoluble in aqueous phase and hence are not available towards hydrophilic microorganisms. Addition of surfactants to enhance effectiveness bioremediation on contaminated soil has been investigated in order to increase the bioavalaibility of several organic contaminants. In Bioremediation, the use of slurry bioreactor has received more attention because it requires a relatively small space and exhibit faster rates of biodegradation when compared with others biotechnological systems. The aim of this research was to improve the bioremediation of a contaminated soil in through the addition of surfactants. For this purpose, two anionic surfactants were investigated: a chemical and a biological one (rhamnolipids). Concentrations of those surfactants were based on their active ingredient and varied on 0.15% up 1.5% w/w. Assays were conduced weighting 300g of dried soil in a cubic flasks with 600 cm3 capacity. Soil samples were mixed with water (50% of field capacity), nutrient enrichment and surfactants on varied concentrations. Experiments were monitored for 60 days, in terms of Oil and Grease (O&G) removal. It was verified that the biodegradation was more effective, after 60 days, with adding biological surfactant (30% of O&G removal) in the concentration of 0.6 % of active ingredient (rhamnolipids). Addition of chemical surfactant as an auxiliary to the bioremediation process increased biodegradation in terms of O&G removal for the concentrations between 0.3% up 1.5%, but no further was observed as a consequence of addition of 0.15% w/w of active ingredient, in relation to the bioestimulated control (5% of O&G removal). After 30 days it was verified a decrease of O&G removal for all experiments. Within the first 30 days the best result was obtained with 0.3% of chemical surfactant (23% of O&G removal) while to the 60 days, this same condition presented 9.5% of O&G removal. Key –words: Biosurfactant, chemical surfactant, petroleum, bioremediation, soil 1. INTRODUCTION Oil pollution from industrial sources, shipping operations and others activities cause a great environmental impact to terrestrial and marine ecosystems. The contamination of soil by hazardous organic pollutants is a current environmental problem. Various approaches have been proposed for treating petroleum hydrocarbon contaminated soil. Among these treatments, the bioremediation technologies have been shown to be a promising technology wherein microorganisms are stimulated to metabolize the crude oil adsorbed in soil matrix (Nano et al. 2003; Sabaté et al. 2004 ). The biological treatment of an oil-contaminated soil can be affected by hydrocarbons structure and weathering processes, which decrease the bioavailability of pollutants to microorganisms. Weathering refers to the result of biological, chemical and physical processes that can affect the type of hydrocarbons that remain in soil (Troquel et al. 2003). Those processes also enhance the sorption of hydrophobic organic contaminants to the soil matrix, decreasing the rate and extent of biodegradation (Bosma et al. 1997). Moreover, a weathered oil-contaminated soil normally contains a recalcitrant
fraction of compounds composed, basically, of high molecular weight hydrocarbons (higher than C25 compounds), which could not be easily degraded by indigenous microorganisms. In contrast, a recently oil-contaminated soil contains a higher amount of saturated and aliphatic compounds, which are the most susceptible to the microbial degradation. Many studies have been demonstrated that the additions of surfactant in the bioremediation process may enhance the oil mobility and increase its availability, improving the biodegradation rates (Alexander, 1994; Kim et al. 2001; Rahman et al. 2003). Surfactants are surface-active agents composed of amphipathic molecules consisting of hydrophilic polar head moiety and a hydrophobic nonpolar tail moiety. They reduce the surface tension by forming an extremely small aggregate that is called micelles. At low concentrations, surfactants are soluble in water, and when increasing their concentration, the molecules form micelles in solution. The smaller concentration at which micelles begin to form is called as the Critical Micelle Concentration (CMC). Above the CMC value, the surfactants have been reported to solubilize petroleum hydrocarbons in soil-water systems, increasing the biodegradation rates (Aronstein & Alexander, 1992; Alexander, 1994; Deshphande, 1999). 2. OBJECTIVE The aim of this work was evaluate the influence of addition of two different anionic surfactant in the treatment of petroleum contaminated soil. 3. MATERIALS AND METHODS 3.1. SOIL SAMPLE Contaminated soil was extracted two years after an oil spill accident occurred in São Paulo (Brazil).. Soil was homogenized, then a representative sample was taken, and further stored at ± 4° C to avoid natural degradation. The chemical and physical properties of the soil are given in Table 1.
Table 1. Chemical and physical properties of contaminated soil Parameter
Soil content
Parameter
Soil content
C organic
46g/kg
pH
5.1
N
1.0 g/Kg
Plasticity
38 %
P
0.001g/Kg
TPH
22 mg/g
K
0.084g/Kg
HPA’s
4.4mg/g
Organic matter
13.7 %
O&G
3.3 %
Sand
59 %
Field Cap. - F/C
38. 5 %
Clay
24 %
Silt
19 %
3.2. SURFACTANTS Two different anionic surfactants were chosen to be evaluated for these experiments: a chemical and a biological (rhamnolipids) one. Concentration range of these surfactants applied to the contaminated soil was based on active ingredient corresponding: 0.15 up 1.5 % w/w (in relation to soil mass) as shown in Table 2.
3.3. EXPERIMENTAL SET-UP Into a glass cubic flask of 600 cm3 capacity, were added 300g of dried soil. Soil samples were neutralized with Ca(OH)2 and then supplemented with nutrients based on optimum nutritional ratio C:N: P = 100:2,5:1. This ratio, had been established in others essays as excellent for this soil biodegradation (Trindade et al, 2005). Enrichment with nutrients was made through the addition of KH2PO4. Humidity was adjusted to 50% of soil Field Capacity. Assays were incubated at 30°C, during 60 days and periodically checked for pH analysis, aeration. When shown necessary, new correction of nutrients, pH and humidity were done. Surfactants for each experiment were added as shown in Table 2. Table 2: Conditions used in bioassays with the addition of surfactants (that contained 300g of soil, correction of nutrients (C:P = 100:1), pH = 7 and humidity (50% of soil the field capacity)): Conditions
Concentration of chemical surfactant
Concentration of biological surfactant
Control
-
-
1
-
0.15% p/p
2
-
0.3% p/p
3
-
0.6% p/p
4
-
0.9 p/p
5
-
1.5% p/p
6
0.15% p/p
-
7
0.3% p/p
-
8
0.6% p/p
-
9
0.9 p/p
-
10
1.5% p/p
-
3.4. ANALYTICAL METHODOLOGIES 3.4.1. Apparent Critical Micelle Concentration (ACMC) and Critical Micelle concentration (CMC) Measurements on surface tension were carried out accordingly in different dilutions of surfactants in a Soil-Water-Oil (SWO) systems. Surfactant solutions had been mixed in a ratio 1:10 (soil:solution). These mixtures were agitated in a shaker during 16 h. After agitation, mixtures were centrifuged at 3000 rpm during 10 minutes. Supernatants were filtered to verify only the solubilized oil after surfactant applications. Surface tension was measured using ring method in a digital-Tensiometer K10T (Kruss, Hamburg, Germany). The ACMC value expressed in mg/L was obtained from the plot of surface tension versus surfactant concentrations solution. 3.4.2. pH Determination 20g of soil was removed from each experiment, in interval of 30 days, and mixed with 50mL of distilled water. After that, the samples were agitated in a shaker during 1h and then the pH value was measured.
3.4.3. Oil and grease (O&G) determination Oil and grease determination was quantified by gravimetric method, extracting 2g of dried soil with hexane by ultrasound (Rizzo & Raimundo, 2003). 3.4.4. Quantification of microbial population Quantification of the final and initial microbial population was made by the pour plate technique. The results were expressed in terms of Counting Forming Unit (CFU)/g of soil (Uruhay, 1998). 4. RESULTS AND DISCUSSION 4.1. EVALUATION OF THE BIODEGRADATION BY O&G REMOVAL Figure 1 shows the results of oil and grease (O&G) removal in the bioassays when it had been added a biological surfactant as well as the results of biodegradation of the biostimulated and the sodium azide control (abiotic control). It was verified that, after 60 days, the highest O&G removal was obtained when 0.6 % of rhamnolipid was applied (30% of O&G removal). Except to the experiment using 1.5% (w/w) of rhamnolipids, all others condition tested had shown a tendency to reach greater oil removal above 60 days. In those assays the results of biodegradation varied of 19.2 up to 30%, being results very above of the biostimulated (5%) and the azide (0.8%) control ones, proving the effectiveness of the biosurfactant addition. 35
0.15% 0.3% 0.6% 0.9% 1.5% Control azide control
O&G removal (%)
30 25 20 15 10 5 0 0
10
20
30
40
50
60
70
Time (days)
Figure 1: Evaluation of O&G removal (%) after the addition of biosurfactant in different concentrations On the same way, Figure 2 shows results on O&G removal in the bioassays in which it was added a chemical surfactant. It was observed, after 30 days, that there was a decrease of oil and grease removal for all experiments. Until the first 30 days the best result was obtained when 0.3% of chemical surfactant was applied (23% of O&G removal). However, at the end of the experiment (after 60 days)it dropped until a level of 9.5% of O&G removal.
0.15% 0.3% 0.6% 0.9% 1.5% control azide control
25
O&G removal (%)
20
15
10
5
0 0
10
20
30
40
50
Time (days)
60
70
Figure 2: Evaluation of O&G removal (%) after the addition of chemical surfactant in different concentrations Results presented in Figure 3 (A and B) confirm which were the best conditions with the addition of surfactants (biological and chemical one), after 30 and 60 days. It was observed on Figure 3A that in first 30 days of biodegradation, the higher was the biosurfactant concentration, the highest was the oil and grease removal. However, after the 60 days, it was an optimum concentration, as already previously, said (0.6% of rhamnolipid concentration). It was still observed, after 60 days, experiments, using 0.9 % and 1.5% had a decrease of O&G removal in relation to the best condition (0,6% of rhamnolipid). A decrease of O&G removal of these experiments could be associated to an extreme agglomeration of the biosurfactant with the soil matrix. Maybe, this extreme agglomeration of the biosurfactant could contribute for reduction of the oxygenation for the aerobic microorganisms, decreasing the effectiveness of the biodegradation process. Another factor should be considered is that high biosurfactant concentrations could be toxic to the biodegradation system. The Figure 3B shows that the best condition was the 0.3% after 30 days, as previously said. 35
25
23
30
30
30 days 60 days
25,5
25 20 15 30 days biological 60 days biological
10
O&G removal (%)
O%G Removal (%)
20
15 9,5
10
5
5 0 0
0,2
0,4
0,6
0,8
1
surfactant (%)
A
1,2
1,4
1,6
0 0
0,5
1
1,5
chemical surfactant concentration (%p/p)
B
Figure 3: The best condition on 30 and 60 days after the addition of the biosurfatant (A) and the chemical surfactant (B).
2
4.2 – EVALUATION OF CMC AND ACMC IN RELATION TO THE OIL AND GREASE REMOVAL Analyzing the two surfactants employed, the chemical surfactant had an ACMC value greater than the biological one, as can be seen in Table 3. These characteristics of biosurfactants to have low ACMA and /or CMC values have being investigated for some authors (Stelmack et al., 1999; Banat, 1995). The CMC value for these different surfactants can varies so much with their structure, but others factors like the temperature of the solution and the presence of organic compounds can affect the CMC value (Haigh, 1996). Surfactants that have low CMC and ACMC value are more appreciable because they can be used in a fewer amounts in relation to others with high CMC and ACMC values, to get similar effect. ACMC value was much higher than the CMC value for both surfactants investigated in the soil-water-oil (SWO) system (Table 3). In the SWO systems the CMC value is modified because there are an extensive adsorption of surfactants onto soil surfaces, which means that much more surfactants are required in soil that might be expected to reach the CMC in systems utilizing clear water (Haigh, 1996). The ACMC values of chemical surfactant were above the biological one. ACMC and CMC values are important, since the addition of surfactant was evaluated in relation to the active ingredient and not on the ACMC value. The ACMC values of chemical surfactant were above the biological one. So, the chemical surfactant should be added in these bioassays in a bigger concentration than the biological one (about 5 times, in accordance with the ACMC values- Table 3), increasing most probably operational cost. Table 3. CMC and ACMC values for each surfactant Surfactant
CMC (mg/L)
ACMC (g/L)
Chemical
100
5
Biological
43
1
4.3 – EVALUATION OF MICROBIAL POPULATION IN RELATION TO THE OIL AND GREASE REMOVAL It were observed in Table 4 that the sodium azide control (abiotic control) did not present microorganisms population in the soil, along the 60 days, as was expected. Biostimulated control kept the same order of microorganisms population magnitude along the 60 days. Experiment with 0.3% w/w of chemical surfactant and 1.5% w/w of rhamnolipids decrease in microorganism population after 60 days (in relation to the 30 days) and this had also reflected in the efficiency of O&G removal. These results are in accordance with those previously presented in Figure 1, which indicated a decrease in O&G removal after 60 days of biodegradation process. In the experiment with 0.6% of rhamnolipid occurred an increase in microorganisms population after 30 days remaining the same magnitude after 60 days, indicating that these microorganisms could biodegrade more oil above the 60 days, as shown in Figure 1. Table4: Microorganisms population of the best condition MICROORGANISMS (UFC/g SOIL) t=0
t=30 days
T = 60 days
Azide control
0
0
0
Control
1.4x106
4.6 x106
3.4x106
0.3% (chemical)
3.2x106
2.5x106
4.5x105
0.6% (biological)
1.6x106
6.1x108
3.0x108
1.5%(biological)
2.8x106
2.1x107
2.5x106
CONDITIONS
4.6 – EVALUATION OF pH ALONG THE 60 DAYS OF BIODEGRADATION During the biodegradation process all the experiments had been monitored by pH value and humidity, so as to keep humidity in 50% of the capacity of field and pH near 7.0. Figure 5 shows profiles for pH with addition of chemical surfactant (A) and biosurfactant (B). It can be observed in Figure 4A and 4B that it was not necessary to adjust the pH along the 60 days, since the pH values were kept between 6 and 7.4 during the biodegradation process.
A
8 7 6 5
0 day 30 days 60 days
pH
4 3 2 1 0 0.15
0.3
0.6
0.9
1.5
% chemical surfactant
9
B
8 7
0 days
6
pH 5
30 days
4 3
60 days
2 1 0
0.15
0.3
0.6
0.9
1.5
Biosurfactant concentration (p/p)
Figure 5. Soil samples pH with additions of chemical surfactant (A) and biosurfactant (B) 5 . CONCLUSIONS Additions of a biosurfactant as an auxiliary to the bioremediation process increased the biodegradation in terms of oil and grease removal for all concentrations added. This improvement can be seen in relation to the biostimulated control (without addition of biosurfactant), whose biodegradation rates was 5%. Biodegradation measurement for all experiments which have addition of biosurfactant varied between 19.2 up 30 % of O&G removal after 60 days. Addition of a chemical surfactant as an auxiliary to bioremediation process increased the biodegradation in terms of oil and grease removal when using concentration between 0.3% up 1.5% w/w, but no further was observed in addition of 0.15% w/w of active ingredient, in relation to the bioestimulated control. After 30 days it was verified a decrease of oil and grease removal for all
experiments. After 30 days the best condition was 0.3% of chemical surfactant (23% of O&G removal) and after 60 days, this same condition presented 9.5% of O&G removal. After 60 days of biodegradation process the best condition was the addition of 0.6% of rhamnolipids in the soil . This experiment presented tendency to increase the biodegradation rates above 60 days , as well as the tendency in increasing the microorganism population. Determinations of ACMC and CMC values were necessary to know the surfactants characteristics and the best conditions to use them. Great additions of surfactant are necessary when surfactants have high CMC and/or ACMC values, in comparison with other surfactants which have lower values. 6- ACKNOWLEDGEMENTS This work was supported by Center of Mineral Technology (CETEM) in partnership with CENPES – Petrobras. The authors wish to thank Ms. Marisa Monte and Antonieta Middea (CTM/CETEM) for the CMC and ACMC tension analysis and Luiz Sobral for the very rich discussing regarding this paper. 7- REFERENCES Alexander, M. Biodegradation and Bioremediation. Academic Press. California. 302p. (1994). Aronstein, B.N. and Alexander, M. “Surfactants at low concentrations stimulate biodegradation of sorbed hydrocarbons in samples of aquifer sand and soil slurries”, Environ. Toxicol. Chem. , 11, 1227-33. (1992). Banat, I.M. ‘Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: A review”. Bioresource Technology, 51, 1-12. (1995). Bosma TNP, Middeldorp PJM, Schraa G & Zehnder AJB. Mass transfer limitation of biotransformation: quantifying bioavailability. Environmental Science & Technology, 31(1): 248252. (1997). Deshpande, S. Shiau, B.J.; Wade, D.; Sabatini, D.A. and Harwell, J.H. “Surfactants selection for enhancing ex situ soil washing”. Pergamon., 33 (2), 351-60. (1999). Haigh, S.D. “A review of the interaction of surfactants with organic contaminants in soil”. The Science of the Total Environment, 185, 161-70. (1996). Nano, G.;Borroni, A. Rota, R. Combined slurry and solid-phase bioremediation of diesel
contaminated soils . Journal of Hazardous Materials, 100, 79-94. 2003. Rahman, K.S.M., Rahman, T.J.; Kourkoutas, Y. Petsas, I. Marchant, R. Banat, I.M. Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipids and micronutrients. Bioresourse Technology, 90, 159-168. 2003
Rizzo, A.C.L; Raimundo,R. Determinação de óleos e graxas, em solo, por gravimetria empregando método de extração com ultrassom”. IT2003-001-00 – Instrução de Trabalho elaborada para o CETEM. Sabaté, J. Viñas, M. and Solanas, A. M. Laboratory-scale bioremediation experiments on hydrocarbon-contaminated soils. International Biodeterioration & Biodegradation, 54, 19-25. 2004.
Stelmack, P.L.; Gray, M.R. and Pickard, M.A. “Bacterial Adhesion to soil contaminants in the presence of surfactants”. Applied and Environmental Microbiology, 65, (1), 163-8, Jan. (1999).
Trindade, P. V. O., Rizzo, A. C. L., Soriano, A. U., Sobral, L. G. S. and Leite, S. G. F. Bioremediation of a weathered and a recently oil-contaminated soi ls from Brazil: a comparison study. Chemosphere, V. 58, Issue 4 , January 2005, Pages 515-522. Troquel J, Larroche C & Dussap CG. Evidence for the occurance of an oxygen limitation during soil bioremediation by solid-state fermentation. Biochemical Engineering Journal, 13:103-112.(2003) Ururahy, A. F. P. Biodegradação de Resíduo Oleoso Proveniente de Refinaria de Petróleo. Tese DSc., Universidade Federal do Rio de Janeiro, Escola de Química, Rio de Janeiro, Brasil, 344p. (1998).
