Natural Attenuation and Biostimulation of Biodiesel Contaminated Soils from Sout

Liliane R.R. Meneghetti1, Ant?nio Thomé2, Fernando Schnaid1, Pedro D.M. Prietto2 and Gabriel Cavelh?o2

1. Graduation Program in Civil Engineering, Federal University of Rio Grande do Sul, Porto Alegre 90035-190, Brazil

2. Graduation Program in Engineering, University of Passo Fundo, Passo Fundo 99052-900, Brazil

Received: May 12, 2011 / Accepted: August 11, 2011 / Published: February 20, 2012.

Abstract: Biodiesel bioremediation in soils may occur by natural attenuation or by engineered techniques, such as biostimulation and bioaugmentation. The present study evaluated the degradation of biodiesel in two soils with different particle size characteristics by the bioremediation processes of natural attenuation and biostimulation. The experiment was carried out ex situ, with the factors temperature, moisture content, and pH being controlled for the experimental period of 110 days. The study aimed at evaluating the biodegradation of a clayey soil (A) and a sandy soil (B), both contaminated with pure biodiesel, by using the analytical methods of respirometry and gas chromatography. Biostimulation treatments using nitrogen, phosphorus, and potassium solutions (NPK) promoted higher microbiological activity in both soils. At the end of the experiment, it was observed that biostimulation was more efficient when compared to natural attenuation, showing higher biodiesel degradation for both soils A (59.76%) and B (90.41%).

Key words: Biodegradation, CO2 evolution, gas chromatography.

1. Introduction??

Most energy sources in Earth are derived from oil, coal, and natural gases. However, these sources are finite [1]. During the last few decades, the limited oil supply and excessive carbon monoxide levels in the air of large urban areas have led some countries, including Brazil, to seek alternative energy sources. The use of alcohol as gasoline additive started in the 1930s, which differentiates Brazilian gasoline from that sold in other countries. The use of another alternative energy source became mandatory by Act number 11.097 of the Brazilian legislation, which established the introduction of biodiesel in the Brazilian energy matrix. Today, 5% is the minimal mandatory percentage of biodiesel addition in the diesel oil sold to consumers in Brazil.

These measures reduce the negative environmental impacts in case of contamination, considering that additives from plant or animal origin are more biodegradable. However, soil contamination remains an environmental concern due to the frequency and the intensity of contamination episodes. Numerous leakages, spills, and accidents during fuel transportation and storage have been reported by environmental protection agencies in the last few years [2].

When the soil is contaminated by fossil fuels and their derivatives, several physical, chemical, and microbiological processes can be used as remediation techniques. Bioremediation is one of the most frequently studied microbiological processes in the last few years. Several studies have been carried out on the bioremediation of fuels, including those on gasoline [3-6], diesel oil [6-11] and biodiesel [11, 12].

In the process of bioremediation, pollutant biodegradation occurs by the action of microorganisms present (natural attenuation) or inoculated (bioaugmentation) in the contaminated soil. It may also be stimulated by the addition of nutrients, such as organic matter, oxygen, nitrogen, phosphorus, potassium, etc. [11]. The process of biodegradation is based on the capacity of the microbial population to modify or to break down the pollutant molecules, by using biodiesel as a source of carbon. The complete degradation of the resulting fatty acids generates non-toxic final products, including carbon dioxide(CO2), water (H2O), and biomass. Ref. [13] also shows that soil microorganisms have extensive catabolic actions, and that a simple way of eliminating pollutants is to add compounds or materials to the soil to stimulate the indigenous microflora growth.

The objective of the present study was therefore to evaluate the biodegradation of two soils with different particle size distributions contaminated with pure biodiesel, by using two bioremediation processes: natural attenuation and biostimulation.

2. Materials and Methods

2.1 Soils

The physical and chemical properties of the two soils studied in the present work are shown in Table 1.

Soil A is a clayey residual soil (horizon B) derived from a basaltic rock belonging to the Serra Geral Formation [14-17]. The samples were retrieved from a cut slope at the Experimental Geotechnical Field of the University of Passo Fundo, located in the city of Passo Fundo/RS, in southern Brazil. Soil B is described as a silty-sandy residual soil (horizon C) derived from a sandstone rock belonging to the Botucatu Formation. Samples were obtained from a cut slope at the side of the RS-240 state highway, located in the city of S?o Leopoldo/RS, also in southern Brazil.

Particle size properties were obtained from a particle-size test carried out according to NBR 7181[18]. Specific gravity was determined according to NBR 6508 [19] and consistency limits (LL, LP) followed NBR 6459 [20] and NBR 7180 [21]. Chemical properties were determined according to the methods described by Tedesco et al. [22]. Fig. 1 shows the particle size curves obtained for soils A and B.

Fig. 1 shows that soil A consists of 68% clay, 7% silt, 22% fine sand, and 3% medium sand, whereas soil B consists of 6% clay, 28% silt, 60% fine sand, and 6% medium sand. When contaminated with oil, clayey soils may present limited efficiency in the process of biodegradation due to their physical characteristics. On the other hand, in basalt residual soils (oxisol) presenting adequate structure, the amount of macroporosity is high enough to allow the migration of air and nutrients that favor bioremediation [11].

2.2 Biodegradation Experiments

A total number of 24 deformed soil samples were stored in sealed 2 L glass flasks for 110 days. Each sample consisted of 500 g of soil with final moisture contents of 36.5% for soil A and 13.5% for soil B, considering natural soil moisture plus contaminant moisture. The soil samples were contaminated with pure biodiesel (B100) of vegetable origin at a ratio of 25 mL per kg of dry soil and the NPK solution prepared according to the CO (carbon organic) present in the soil after contamination, adjusting the NPK amount present in the natural soil to a CNPK ratio of 100:10:1:1. This ratio, as described by Ref. [23], is considered optimal for the development of most microorganisms. Also, according to Ref. [24], the CNP ratio required to convert oil carbon into biomass is 100:10:1.

A biostimulating solution of 2.5 mL/kg of dry soil was used for both soils. In soil A, the concentrations applied were 0.275 g NH4NO3/50 mL and 0.875 g KH2PO4/50 mL of distilled water, and in soil B, 0.55g NH4NO3/50 mL and 1.75 g KH2PO4/50 mL. Bento et al. [10] used the same biostimulating solution, but with different nutrient concentrations.

The experimental planning summary is shown in Table 2. Eight treatments were carried out in independent triplicates, resulting in 24 experimental runs. Treatments T1, T2, T3, and T4 corresponded to soil A and treatments T5, T6, T7, and T8 to soil B.

The soil microbiological activity was tested by using the respirometry technique [25-28]. In this method, the CO2 produced in the test is captured by the 20 mL NaOH 0.40 M inserted in a 50 mL glass beaker inside the microcosm. This allows a qualitative, ecological analysis (observation in the natural habitat), by observing both the interactions and inter-relations of microorganisms with soil particles [26]. All tests in this experiment were performed at room temperature.

Contaminant degradation was assessed by using the chromatographic profile obtained for all treatments. Gas chromatography was performed at the end of the each test through a gas chromatographer coupled to a flame ionization detector (VARIAN, model STAR 3400 CX). Degradation percentage was calculated as follows (Eq. (1)).

(1)

Experimental data were submitted to statistical analysis by using the Tukey’s Test for multiple comparisons of means at a 5% probability level.

3. Results and Discussion

3.1 CO2 Evolution

The CO2 release is a factor that determines the efficiency of bioremediation processes. Mineralization studies involving the evaluation of total CO2 production generate excellent information of the biodegradation potential of hydrocarbons in contaminated soils [29]. The microbiological activity of the investigated treatments obtained by the release of CO2 accumulated during 110 days is presented in Fig. 2.

In Fig. 2, an initial microbial growth up to 30 days can be observed for all treatments, which might be attributed to the adaptation of the microorganisms to the soil and to the presence of bioavailable nutrients. Treatments with the addition of both biodiesel and NPK solution presented the highest CO2 accumulation and, consequently, the highest microbiological activity due to the bioavailability of nutrients (CNPK). After this initial phase, treatments T6 and T8 presented higher growth rates relative to the other treatments. At the end of the experiment, T8 presented the highest microbiological activity, with CO2 accumulation of 317.45 mg per kg of soil, followed by treatments T4 and T6 with 196.17 mg/kg and 205.87 mg/kg, respectively. During the investigated period, the CO2 production curves did not reach a plateau, indicating continuous microbial growth.

According to Chenu et al. [30], microbial activity and the rate of biological functions vary according to soil type, which is determinant for the biodegradation of organic compounds. As previously described, the same contamination and biostimulation methodologies were applied to treatments T4 and T8, but T8 presented higher microbiological activity due to the type of soil. Sandy soils, such as soil B, present higher porosity than clayey soils (soil A), allowing oxygen diffusion during the biodegradation process. In general, higher oxygen availability produces higher soil microbiological activity. Some clayey soils contaminated with oil have some characteristics that may limit the efficacy of the bioremediation process, including low permeability and low oxygen and nutrient diffusion [31]. In addition, it may be inferred that clayey soils present more affinity and higher contaminant retention on the particles surfaces due to electro-chemical forces, thereby hindering the action of microorganisms.

The statistical analysis of the CO2 evolution as a function of time and bioremediation treatments is presented in Tables 3 and 4.

In Table 3, the comparisons are made through columns, i.e. among bioremediation treatments at a given time, while in Table 4 the comparisons are made through rows, i.e. among times for a given treatment.

Table 3 shows that T8 and T4 presented bioremediation processes with statistically higher microbiological activity than the other remediation processes since the beginning of the experiment. After 30 days, T6 was statistically similar to T4, and different from the other treatments. At day 110, the treatments T1, T2, T3, T5 and T7 were not statistically different.

From Table 4, it is evidenced that T8 was different from the other treatments at all evaluated times, whereas significant differences among the other treatments only appeared at the end of the investigated period. All the investigated factors (bioremediation process, soil and contaminant) and their interactions presented significant effects (p < 0.05) at all analyzed times.

3.2 Gas Chromatography (GC)

At the end of the tests, methyl ester percentages were compared with the methyl ester percentage present in pure biodiesel (control). The results are presented in Table 5.

The chromatographic analysis showed a reduction in the unsaturated fatty acid esters (C18:1 Cis, C18:2 Cis, and C18:3 Cis) for all treatments. On the other hand, the percentages of saturated fatty acids increased (C14:0, C16:0, C18:0, C20:0, and C18:1 T). In treatments T2 and T4, this percentage tripled and in treatments T6 and T8, this percentage was five times higher than in the control treatment. Trans oleic acid(C18:1 T) was not detected in the control treatment, but its concentration increased in the other treatments at the end of the experiment and ranged between 3.06% in T6 and 17.94% in T4. In treatment T4, myristic acid (C14:0) and particularly linolenic acid(C18:3 Cis) were fully reduced as compared to the control treatment, indicating complete degradation of these compounds.

The transformations experienced by fatty acids during the experiment are directly influenced by the degradation of the double bonds of carbon chains. According to Seklemova et al. [32], these bonds are more susceptible to degradation reactions than single bonds.

Fig. 3 shows the comparison between the degradation percentage caused by natural attenuation and biostimulation in soils A and B at the end of the investigated period (110 days).

At 110 days, as shown in Fig. 3, biodiesel was biodegraded by both bioremediation processes in both soils, but soil B presented higher degradation percentage as compared to soil A. Biostimulation degraded up to 90.42% of the biodiesel in soil B (T8)

and 59.76% in soil A (T4), whereas natural attenuation resulted in 58.5% biodiesel degradation in soil B (T6) and 16.8% in soil A (T2).

According to Santos et al. [31], the process of natural attenuation of an organic pollutant is continuous due to the adaptation of natural microorganisms to the soil. These microorganisms start to use the organic compound as a carbon source, thereby reducing its concentration with time. Therefore, natural attenuation, if no nutrients are added or if not adapted to the environmental conditions, requires more time for degradation as compared to other bioremediation processes, such as biostimulation.

The efficiency of the biostimulation process is directly related to the C:N:P:K ratio present in the contaminated soil. Studies showed that the optimal C:N:P:K ratio for the growth of most microorganisms is 100:10:1:1 [33, 34]. Several authors have studied biodiesel biodegradation in the laboratory and have achieved 90% efficiency of diesel biodegradation by the addition of nutrients (NPK) at optimal ratios for microbial growth [34, 35].

The presented results indicated that the bioremediation of an organic compound depends on several factors, such as soil characteristics, nature of the contaminant, and the presence of microorganisms. 4. Conclusions

Based on the results obtained in the present work, it was possible to conclude that:

Biostimulation was the bioremediation technique that presented the highest microbiological activity in

both soils;

The levels of unsaturated fatty acids were reduced for all treatments;

The technique of natural attenuation presented satisfactory results, and it was demonstrated that the time involved in the natural attenuation process is usually longer as compared to other bioremediation processes;

When the results obtained by both degradation methods applied are evaluated at the end of the experiment, it is possible to conclude that higher CO2 evolution is equivalent to higher degradation of biodiesel compounds;

The soil with a silty-sandy matrix presented a better performance when submitted to biodiesel degradation as compared to the clayey soil.

Acknowledgments

The authors wish to express their gratitude to the Brazilian Research Councils (CNPq/MCT, Fapergs, and CAPES/MEC) for the financial support to the research group.

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