Comparative Kinetic Study of Different Bioremediation Processes for Soil Contaminated with Petroleum Hydrocarbons

Bioremediation of hydrocarbon polluted soil can be achieved by natural attenuation, biostimulation and/or bioaugmentation processes. In this study the three technologies were evaluated to treat hydrocarbons polluted soil of total petroleum hydrocarbon (TPH) content of 42,000mg/kg semipilot scale cells, over an incubation period of 120 days at room temperature (25-30°C), moisture content of 45% and pH around neutrality. Bioaugmentation with bacterial consortium Pseudomonas aeruginosa I.1.1.6 and Brevibacterium casei I.2.1.7 showed the highest degradation potential (76%), followed by biostimulation process with biodegradation efficiency of (62%) and then come the natural attenuation (48%). Kinetic modeling was performed to estimate the rates of TPH biodegradation in the studied systems. Three different error functions (root mean square, sum of the absolute errors and average relative error) were employed in this study to evaluate the goodness of fit of the model equation to the obtained experimental data. This showed that the degradation was found to follow first order model. The highest rate constant (0.012 day-1) was observed in cell augmented with bacterial consortium I.1.1.6 and I.2.1.7, followed by biostimulation cell (0.008 day-1). The lowest rate constant was observed in natural attenuation cell (0.005 day-1). Accumulative evaluation of CO2 was good qualitative indicator of biodegradation activity in each cell. The CO2 formations in bioaugmented cells were relatively higher than those in natural attenuation and biostimulation cells.

consequences of the physicochemical methods make bioremediation more attractive and as one of the most successful technology for clean up contaminated sites.
Bioremediation use mainly three strategies (Kaplan and Kitts, 2004); natural attenuation, biostimulation and bioaugmentation. The simplest method of bioremediation to implement is natural attenuation, where contaminated sites are only monitored for contaminant concentration to assure regulators that natural processes of contaminant degradation are active. Biostimulation requires adjustments to contaminated soil in order to provide bacterial communities with a favorable environment in which they can effectively degrade contaminants. This includes the addition of nutrients, adjustment of pH and moisture content while also making appropriate adjustments for the proliferation of indigenous microorganisms, hence speeding up the bioremediation process. In case where natural communities of degrading bacteria are present in low numbers or even absent, bioaugmentation i.e., the addition of contaminants-degrading microorganisms, can speed up the degradation process.
We have isolated several candidate bacterial strains , Farahat et al, 2006and Rafaat et al, 2007 in our effort in developing an active bacteria consortium that could be of relevance in the bioremediation of crude oil contaminated systems in Egypt. Three of these bacterial isolates, Pseudomonas synxantha I. This paper reports a comparative kinetic study was performed to provide necessary information for the possible bioremediation of oil polluted soil using these three bacterial isolates compared to biostimulation and natural attenuation processes in a semi-pilot scale.

Polluted soil
Oil polluted soil of total petroleum hydrocarbon content of (42,000mg/kg) was collected from a drilling site of an oil field in Suez Gulf, Egypt.

Bacterial strains
Pseudomonas synxantha I.  (Kirimura et al., 2001) was used to obtain biomass for augmenting the polluted soil.

Bioremediation treatments
The remediation study took place over a period of four months at room temperature (25-30 o C), pH around neutrality and moisture content of the soil was about 45%. The three biotreatment processes are illustrated as follows:

Biostimulation
10 kg of oil polluted soil were placed in plexi-glass cell (cell 1) of dimensions ( 60 X 60 X 20 cm) supplied with an air sparger connected to an air compressor of capacity ( 200-300 L /min), as illustrated in Fig 1. The cell was incubated at room Nutrients: NH 4 Cl and K 2 HPO 4 were added to keep the ratio of C:N:P (100:10:1).
Periodical tilling and addition of water were done day after day to ensure good aeration and to keep the moisture content around 45% in addition to assuring good dispersal of microbial population, nutrients and surfactants.

Bioaugmentation
This treatment was equivalent to biostimulation treatment with the exception of adding bacterial isolates in the following criteria:

Natural attenuation
A plexi-glass cell (cell 6) containing oil polluted soil without addition of nutrients or bacteria.
Periodical tilling and addition of water were done day after day to ensure good aeration and to keep the moisture content around 45%.

Monitoring of bioremediation TPH concentration
Gravimetric determination of TPH concentration was done according to the method described by Viguri et al., (2002).

CO 2 evaluation
Accumulative production of CO 2 as a measurement of soil heterotrophic activity was done according to the method described by Isermeyer (1952).

Kinetic modeling
This was preformed to estimate the rates of TPH biodegradation in the studied systems. Kinetics of reaction can be described in terms of its order (Sarkar et al., 2005). First order kinetic model: ... (1) Second order kinetic model: ... (2) where C is the TPH concentration (mg/kg), t expresses time, K 1 and K 2 are the first and second order rate constants, respectively where A and B are constants.

TPH removal
The most direct way to measure bioremediation efficiency is to monitor hydrocarbon disappearance rates (Margesin and Shinner, 2001). In this study, bioaugmentation with different bacterial consortia produced a significant impact on the removal of TPH. The biodegradation efficiencies in cells augmented with bacterial consortia were better than that augmented with individual bacterial isolate in the following order cell 4 > cell 3 > cell 5  cell 2.
The advantage of employing mixed cultures as opposed to pure cultures in bioremediation has also been widely demonstrated (Ghazali et al., 2004). It could be attributed to the effects of synergistic interactions among members of the association. Mechanisms through which bacteria benefit from synergistic interactions are complex. It is possible that one species removes the toxic metabolites (that otherwise may hinder microbial activities) of the species preceding it. It is also possible that the second species are able to degrade compounds that the first are not able to degrade or partially degrade them (Alexander, 1999).

Degradation rates of TPH
Kinetic modeling was performed to estimate the biodegradation rates in the studied systems.
To determine the order of the reactions in each of the soil treatments, the data were plotted in a scatter diagram. Fig. 3 and 4 represent the data plots of first order and second order kinetics models, respectively. All the parameters obtained for the two models are presented in Tables 1 and 2, respectively.   Natural attenuation 2x10 -7 0.970 The R value represents the correlation coefficient of the data, the nearer the value of R to 1, the stronger the correlation of the data (Everitt, 2002). The obtained R values for the plots in all the studied systems were in the range 0.904-0.967 for first order and 0.929-0.977 for second order models. A relatively high and close R values indicated that both models successfully described the kinetics of the degradation of petroleum hydrocarbon components.

Error analysis for the studied kinetics
Since both first order and second order rate equations gave close R values, error analysis were necessary to differentiate between the two models. Three different error functions were used to determine how well models represent the experimental data. The error functions employed were as follows:   Where N is the number of data points, C cal is the calculated data from the kinetic models and C exp is the experimental data.
The values of all three error analysis were presented in Table 3; the lower the values of error analysis the better will be the goodness of fit. In the present study, experimental results were better described using a first-order kinetic model. Nocenteni et al., (2000) and Namkong et al., (2002) were adequately described their hydrocarbon degradation data using a first-order kinetic model.

Rate constant and t 1/2
The rate constants are reflective of the relative effects of various treatments on TPH degradation in contaminated soils (Sarkar et al., 2005). The rate constants K 1 obtained from our experimental results of all the treated systems are listed in Table 1.
The highest rate was observed in cell(4) augmented with bacterial consortium I. 1 The half-life of TPH biodegradation listed in Table 1 confirmed the experimental results illustrated in Fig. 1

Heterotrophic activity (CO 2 )
CO 2 evolution was used as a measure of soil heterotrophic activity. In Fig. 6 the profile of CO 2 evaluation of the six experimental sets are shown: Overall there were considerable variations in CO 2 evolution trends between studied systems. These variations indicated that CO 2 measurements were good qualitative indicator of biodegradation activity in each cell. It can be observed that natural attenuation showed the lowest CO 2 production (178003 mmol/kg soil) after 120 days of incubation. Generally biostimulation and bioaugmentation processes caused increase in the heterotrophic activity of the treated cells. Similar observations were reported by Nocentini et al., (2000).
The highest CO 2 production was observed in cell 3 bioaugmented with Pseudomonas aeruginosa I.1.1.6, where (312470 mmol/kg soil) of CO 2 was recorded after 120 days of incubation. Other cells showed intermediate production of CO 2 in the following order cell 3> cell 4  cell 5  cell 1, where 252520, 242460, 236490 and 233050 mmol/kg soil of CO 2 were recorded, respectively after 120 days of incubation.
In all cells CO 2 production rate was high during the first 40 days of incubation followed by a stationary phase up to 70 days of incubation then the production rate increased again up to 90 days of incubation then followed by another stationary phase up to 120 days of incubation.
According to Sabate et al., (2004), the differences in CO 2 evolution could be attributed to the absence of assemble sources of carbon and energy or to a presence of toxic compounds.
The CO 2 formation rates in bioaugmented cells were relatively high than natural attenuation and biostimulation cells. This fact would suggest that some positive effects of our augmented bacterial isolates in reclamation of oil polluted soil.