evaluation of the biostimulation of crude oil

2nd Mercosur Congress on Chemical Engineering 4 Mercosur Congress on Process Systems Engineering th

EVALUATION OF THE BIOSTIMULATION OF CRUDE OIL CONTAMINATED CLAY SOIL Sandro J. Baptista*, Magali C. Cammarota, Denize D. Carvalho* Escola de Química - Universidade Federal do Rio de Janeiro

Abstract. Biostimulation is a technique that has been used as way to adequate the environmental conditions in order to stimulate the microbial activity in the impacted soil, either by adding nutrients and/or terminal electron acceptors, or adjusting soil pH and/or moisture content. Optimization of these parameters has been fundamental for bioremediation process of crude oil in the impacted soil. The main target of the present work was to evaluate the biostimulation as an alternative technique that may be able to treat crude oil contaminated clay soil. An experimental design with two-level factors (2k) was performed to achieve the best nutrient contents which would allow the microbial activity of soil to develop and augment in microcosm assays. According to the best results obtained in microcosm assays, the medium was supplemented with 2.5% w/w (NH4)2SO4 and 0.035% w/w KH2PO4 and the experiments were conducted at room temperature and with an air rate of 6 L/h. These biodegradation assays were conducted using an aerobic fixed bed reactor, containing 300g of contaminated soil during a 45-days experiment. To monitor the crude oil biodegradation, the organic matter content (OM), the oil and grease content (O&G) and the total petroleum hydrocarbons content (TPH) were measured. In order to assess the microbial activity in the soil, the CO2 production together with total heterotrophic and hydrocarbon-degrading bacteria count was monitored. It was noticed after 45-days experiment that efficient removals of OM, O&G and TPH were respectively 43%, 34% and 42%, but on the other hand, the removals were below 5% in the control assays. Keywords: Bioremediation, Crude Oil and Clay Soil.

1. Introduction Bioremediation is the application of microbial processes to convert environmental contaminants into harmless substances (for instance, CH4, CO2, H2O). It has emerged as a good technique for environmental treatment regarding organic compounds, such as petroleum hydrocarbons, due to its flexibility and adaptability in different sites (Ryan et al., 1991). The advantages on the study of bioremediation in crude oil contaminated soil treatment is its cost-effectiveness when compared to some physicochemical techniques. However, there are some factors that influence the bioremediation process and should be monitored. These factors include soil structural and textural characteristics, temperature, pollutants type and concentration, nutrients and oxygen availabilities and microorganisms' concentration on the impacted site. Therefore, there is a need to adjust some environmental conditions such as improving soil aeration, monitoring and correcting the moisture and pH in order to stimulate the native microorganism activity and to obtain the best pollutant removals. The physical properties of the organic pollutants that inhibit biodegradation are frequently related to their bioavailability to the microorganisms, that will directly affect microbial activity. Nutrient addition has enhanced the biodegradation of petroleum hydrocarbons in contaminated soils due to their stimulated native microorganisms (Cunningham and Philp, 2000). Nitrogen and phosphorus are the most important limiting nutrients that have been used in biostimulation process in order to support microbial growth

*

To whom all correspondence should be addressed. Address: Escola de Química, UFRJ - Centro de Tecnologia, Bl.E, 21949-900 Rio de Janeiro – Brazil E-mail: [email protected], [email protected]


(Liebeg and Cutright, 1999). However, in some cases, the addition of nutrients may also repress the biodegradation of contaminants, because it could affect microbial activity negatively. The main objective of the present work was to evaluate biodegradation of petroleum hydrocarbons in clay soil, supplemented with nitrogen and phosphorus in order to stimulate native microorganisms.

2. Material and Methods 2.1. Experimental Apparatus Microcosm Assays The contaminated soil was collected and stored at 4ºC. The experiments were held in aerobic fixed bed bioreactors (Figure 1) with 60g of contaminated soil at 35ºC and aeration rate of 6L/h during 30 days. At first, an experimental design 22 was performed to achieve the best nutrient contents, (NH4)2SO4 and KH2PO4, which would allow the microbial activity of soil to develop and increase. The (NH4)2SO4 and KH2PO4 contents were studied 0-5 g /100g soil and 20-50 mg/100g soil, respectively, in duplicated experiment. After experimental design, new assays were performed in order to study different contents of KH2PO4 - 35, 60, 70 and 80 mg/100g soil – without adding nitrogen source ((NH4)2SO4). These assays were held in aerobic fixed bed bioreactors as shown in Figure 1 and performed in duplicated experiment.

Fig. 1. Scheme of an Aerobic Fixed Bed Bioreactor in Microcosm Assays.


Macrocosm Assays The biodegradability experiments were held in four aerobic fixed bed bioreactors (Figure 2), with 300g of contaminated soil at room temperature and aeration rate of 6 L/h, during 45 days-experiments. According to the best results obtained in microcosm assays, the medium was supplemented with 2.5% w/w (NH4)2SO4 and 0.035% w/w KH2PO4. In order to verify the crude oil biodegradation kinetic, the organic matter (OM) content and pH were measured each five days. The monitoring of oil and grease (O&G) and total petroleum hydrocarbon (TPH) contents were performed at 0, 5, 25 and 45 days of experiment. In addition, CO2 content was measured during 45 days and total heterotrophic (BHT) and hydrocarbon-degrading bacteria (BHc) count were also determined in the beginning and after the 45-days experiment. The bioreactors were completely sealed in order to avoid CO2 leakage to the environment before passing through each CO2 removing tubes. In each of them a solution containing Ba(OH)2, aimed at collecting CO2 evolved from biodegradation was added. Thus, all CO2 measured in the bioreactors has actually come from the biodegradation process. Figure 2 shows the set of the experimental apparatus for CO2 dosage, consisting of a bottle of NaOH solution (60%), in order to remove CO2 from the incoming air, a bioreactor and tube series with Ba(OH)2 (0.1M).

Fig. 2. Scheme of an Aerobic Fixed Bed Bioreactor for Collecting CO2 Evolved from Biodegradation.

2.2. Physicochemical Analysis and Bacterial Count The organic matter content (OM) was measured by using the Walkey and Black Method (Jaramillo, 1996), while for oil and grease content (O&G), an adaptation of the methodology described in section 5520 D of the Standard Methods (APHA, 1992) was applied. Total petroleum hydrocarbons (TPH) were extracted with solvent S-316 and measured in infrared equipment OCMA-350 (Horiba, 1995). Nitrogen content in the contaminated soil was measured using the Kjeldahl method and phosphorous by using the total phosphorous method (Jaramillo, 1996). Soil pH was determined by the Jaramillo’s method (1996). In order to determine the soil’s water content, a thermogravimetric balance (IV 2000 GEHAKA) was used. As for


the water-holding capacity of soil, the measurement has been performed using the method described by Santos et al. (2001). CO2 content was measured through the modified version of the method of the Gledhill’s Test in a closed system. The analysis was adapted for each type of tested soil (IBAMA, 1988). Total heterotrophic bacteria was counted according to the spread-plated method on Tryptic Soy Agar (TSA) medium and the results were expressed in colony-forming units (CFU) per gram of dry weight. Hydrocarbon-degrading bacteria (BHc) count was performed according to the Most Probable Number method (MPN) (Volpon et al., 1997) and the unit was expressed as MPN/g of dry weight.

3. Results and Discussion 3.1. Soil Characterization The main physicochemical and microbiological characteristics of the contaminated soil were evaluated before assays. The contaminated soil presented high carbon content levels (44,8±0,2 g/Kg), due to the presence of crude oil in the soil, which in fact was related to oil and grease (O&G) and total petroleum hydrocarbon (TPH) content, 27.5±1.5 g/Kg and 9.7±0.1 g/Kg, respectively. According to The Dutch Target and Intervention Values, a soil needs intervention when TPH contents exceed 5g/Kg, in a soil containing 25% of clay and 10% of organic matter content (Johnson, 2003a). For the Danish (Zealand) Guidelines, the soil can be regarded as contaminated when TPH (C5-C35) content exceeds 0.2g/Kg (Johnson, 2003b). Therefore, the investigated soil presented high TPH levels by all the International Standards evaluated. The soil in question presented neutral pH (7.5±0.1), being thus suitable for contaminant biodegradation by indigenous microorganisms. The moisture content was 53.6±0.3% and could influence biodegradability negatively, as a good soil aeration would be difficult to attain. In order to amend the moisture content, the soil was dried at room temperature up to its 50% water-holding capacity. According to the Jaramillo’s method (1996), the soil tested was rich in nitrogen and poor in phosphorus, so that it had to be supplemented on nutritional shortage, by adding (NH4)2SO4 and KH2PO4. As far as soil microbial population is concerned, representative values for crude oil biodegradation could be verified when compared to the results of Jorgensen et al. (2000). Total heterotrophic and hydrocarbon-degrading bacterial of 8.2x107 CFU/g and 5.4x104 MPN/g, respectively, which could degrade n-alkanes from 50 to 90% of n-alkanes after 5 months was verified. 3.2. Microcosm Assays In the experimental design with two-level factors (2k) the independent variables studied were (NH4)2SO4 and KH2PO4 content. The assays were conduced in function of the organic matter removals (dependent variable) after 30-days experiment at 35ºC. The OM removals were approximately 15% in the bioreactors with 20 mg/100g KH2PO4 and without (NH4)2SO4 content. The same results were obtained in the bioreactors with 20 mg/100g KH2PO4 and 5 g/100g (NH4)2SO4 content. The best results were obtained when the bioreactors were


only supplemented with 50 mg/100g KH2PO4 content, the OM removals were 26%. According to this experimental design, the main variable studied was the KH2PO4 content. In the same way, Liebeg and Cutright (1999) observed in their experiments that addition of phosphorus was more important than the addition of nitrogen in the biodegradation. Part of phosphorus content is adsorbed into matrix soil and this affects its bioavailability for microorganisms (Resende et al., 2002). The contents of (NH4)2SO4 studied were not significant in this experimental design. Figure 3 shows a relationship between OM biodegradation and different phosphorus contents in the bioreactors. It could be observed that the highest OM biodegradation was approximately 35%, with 35 mg KH2PO4/100g and OM removals were decreasing while phosphorus contents increased. The highest phosphorus contents should have inhibited the microbial activity in the contaminated soil (Liebeg and Cutright, 1999).

80 Nitrogen Organic Matter

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Fig. 3. Relationship Between OM Biodegradation and Different KH2PO4 Contents in the Bioreactors.

3.3. Macrocosm Assays

Results of OM degradation kinetic are shown in Figure 4. OM removal efficiency was approximately 16% in 5 days-experiment and a slight decrease was observed after 15 days, because moisture content in the soil was affected by intense variation of the room temperature. According to Li et al. (2000), petroleum hydrocarbon biodegradation is influenced by the interface area between oil and water. After 45-days experiment, OM removals were 42.6%.


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Fig. 4. Monitoring of the Organic Matter Degradation during 45-Days Experiment.

The monitoring of the produced CO2 during 45-days experiment in the aerobic bioreactor was shown in Figure 5. A green curve ( ) plotted on the graph represents the produced CO2 from biostimulated bioreactors and red curve ( ) represents control bioreactors. The curves showed the same profile, but the produced CO2 was 840 mg/kg soil and 350 mg/kg soil in the biostimulated and control bioreactors, respectively, in 5 days. After 45days experiment, the production 3,247 mg/kg soil in the biostimulated bioreactors. A relationship between OM, O&G and TPH degradation efficiencies was shown in Figure 6, after 5, 25 and 45-days experiment. O&G and TPH degradation efficiencies were approximately 20% in 5 days, and 34% and 42% after 45-days experiment, respectively. Results of Figures 5 and 6 showed that the produced CO2 was related to OM, O&G and TPH removal efficiencies, because crude oil degradation was increasing at the same time that CO2 production increased. According to Kao et al. (2001), CO2 production is a by-product of the bioremediation process. Namkoong et al. (2002) also noticed that the TPH degradation was related to microbial respiration and the results were expressed as CO2 production. CO2 production was strongly related to crude oil degradation, because OM, TPH and O&G removal efficiencies, in control bioreactors, were bellow 5% and produced CO2 could be from porous of soil (Prevedello,1996) and/or incoming air.


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Fig. 5. Monitoring Produced CO2 During Biodegradability Assays During 45-Days Experiment. 50

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Fig. 6. Relationship Between Organic Matter, Oil & Grease and TPH Degradation Efficiency During 45-Days Experiment.


In order to evaluate the increase of microbial population in contaminated soil, total heterotrophic bacteria (BHT) and hydrocarbon-degrading bacteria (BHc) were also counted at the beginning of and after 45-days experiment. At the beginning of experiment, the BHT count was 1.8x106 UFC/g and 1.3x107 UFC/g after 45 days. In addition, the BHc count was 1.5x103 NMP/g, at the beginning of experiment, and 4.5x104 NMP/g, after 45 days. On the investigation of bacterial count, it was evidenced that the increase of BHT was related with the increase of BHc in the soil. With regard to soil pH remained neutral during biodegradability assays.

4. Conclusions According to the microcosm assays, the main variable studied was the KH2PO4 content and the highest biodegradation of OM was approximately 35%, with 35 mg KH2PO4/100g soil and OM removals were decreasing while phosphorus and nitrogen contents increased. With regard to the macrocosm assays, it was observed that in 45-days experiment, the efficient removals of OM, O&G and TPH were respectively 43%, 34% and 42%, but on the other hand, the removals were below 5% in the control bioreactors. Although crude oil removals in this work seemed to be low, the results were satisfactory, since the residual petroleum hydrocarbons in the soil investigated were considered recalcitrant, as the soil contamination took place in 2000.

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Acknowledgments The authors wish to thank Agência Nacional de Petróleo (ANP) for sponsoring this research and also Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support.