Coupling In Situ Chemical Oxidation And Bioremediation Technology Biology Essay

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It has been about hundred and eighteen years since Dr. Rudolf Christian Karl Diesel, invented an engine which was going to revolutionize the transportation means world wide. The engine in the beginning, worked with vegetable oil since the petroleum industry by that time was very incipient (Wen Jie, 2009). Over time, the petroleum industry became stronger, and the necessity to produce a fuel similar to fuel-oil to satisfy the performance of the engine in the market was arose. Rapidly, this sort of engine and fuel were improved until 1910, a bit later, when it was applied to massive public transportation, the locomotive (Wikipedia, 2009).

Locomotives like the ones used in a small town in Poland named Węgliniec, where the continuous dumping of diesel fuel on the soil, in a railway refueling station for 30 years (1970 - 2000) (Sutton et al. 2010), becomes an issue for public health. The contaminants carried along by the fuel, reached the groundwater due to seepage coming from the upper layers.

As it threatens the public health, environmental regulations and the need for the wise use of renewable and non-renewable resources in the whole world, different remediation strategies for decontamination of polluted sites with oil products have been developed (Kamnikar, 1992; Hicks and Caplan, 1993; Weyman, 1995).

Remediation Techniques

Diesel fuel is made of a large variety of hydrocarbons (table 1), these can be degraded either under aerobic or anaerobic conditions (Bregnard et al. 1996). Technology offers several options for pollutant degradation in the soil at different levels. They range from the chemical ones to biological, and are described by three components: source, path and the receptor scheme. The most used technologies up to now are excavation and conventional pump and treat (ex situ), but a hopeful alternative to reduce organic contaminants from the soil, is generally ascribed to bioremediation (Upsoil, 2009). Table 2 give us an overview of pro's and con's of the main accustomed techniques.

Although conventional technologies like excavation and pump and treat have been applied ever since, does not mean they are perfect. By taking a look at table 2 we realize that these technologies still have a lot to improve to be the most suitable ones. This is due to the advantage that one technology can have, it is immediately counteracted by the disadvantage(s) that it also has. For instance, excavation fulfils the requirements about working relatively fine in a short time frame, but, it is costly and very destructive for both societal and ecosystems, especially for large polluted sites (Upsoil, 2009). Pump and treat technology applied to polluted water treatment, takes control of contaminant risk and receptor protection, but is not efficient, inasmuch as it takes too long to remediate the bulk of pollutants at the source zone, specially when this is not removed (Upsoil, 2009).

Table 1. Petroleum hydrocarbon composition of diesel fuel (Rodriguez-Martinez, 2006).

Table 2. Current Technologies and aimed-for technologies in the space of optimization for recovery of degraded soils with organic contaminants; green (+): perceived as scoring good on the dimension indicated; (-): perceived as scoring less well (Upsoil, 2009)








Excavation (source zone)




Pump-and-treat (plume)




In situ:

Bioremediation (plume)




Natural Attenuation (plume and source)




Chemical treatments (plume and source)




Aimed for UPSOIL:

Smart coupling of in-situ (source and plume)




Frontier technologies (source and plume)




In situ chemical oxidation technologies have proved to be reliable to diminish the amount of pollutants in the soil in a short period of time. They are very similar to excavation (Upsoil, 2009); nevertheless, the costs of application of this technology can be increased, being that, this is a non-selective technique, and can react with the soil matrix during the process, producing in such way, negative effects in the ecosystem sustainability. This not the case of bioremediation and natural attenuation, for though, they are considered low-cost technologies and achieve the sustainability criteria, they require too much time of aftercare and monitoring periods (Upsoil, 2009).

What has also become visible in table 2 is that, by integrating the approach of in situ chemical oxidation in the reduction of bulk contaminants in areas of high concentration, with a later administration of bioremediation process, it would allow us to treat the remaining low-level concentration of pollutants in a biological way for a longer time frame. By using such approach, the requirements in relation to cost, time, and sustainability aspects, would also be met (Upsoil, 2009). The pro´s and con´s of these two technologies will be exposed as follows.

In Situ Chemical Oxidation by applying Fenton's reagent

In the in situ chemical oxidation (ISCO) approach, chemicals like Sodium permanganate, potassium permanganate or hydrogen peroxide among others, are pumped into the subsurface to interact with the pollutants. Reactions occur inside the soil, being that the treatment takes place in the very same site where the contaminants are. Therefore, the bulk of contaminants will not be removed, posing less environmental risk because there is no need for excavation, transport to treatment facilities or disposal of hazardous waste (Ahlert and Kosson, 1983; Ghassemi, 1988).

A British professor H.J.H. Fenton in the 1890's, described the exothermic and somewhat violent reaction of hydrogen peroxide with iron salts (ferrous sulfate) (Jacobs et al. 2003), improving in this way, the higher oxidative strength of hydrogen peroxide to oxidize a wider variety of contaminants at a faster rate (Jacobs 2003, Upsoil 2009). The name given to this reaction was Fenton's Reagent. Table 3 makes a description of the organic compounds that can be oxidized by applying peroxide-Fenton's reagent and some other.

Table 3. Reactivity of Oxidants (Brown 2003)

Nowadays, this reagent has turned out to be one of the most typically used to treat soil polluted with hydrocarbons. This is due to its relevance in terms of cost-effectiveness (Brown, 2003), which has positioned it as the most important reagent to be applied in ISCO technology. Table 4 shows the relevance in the cost-effectiveness parameter of the peroxide-Fenton's reagent with some other peroxides. From this table can be concluded that, by using Fenton's reagent at either high or low pH, the money invested in treating a certain amount of polluted soil, is even lower in comparison to the other peroxides. This is a very important parameter if we take into consideration, that companies will prefer the less expensive chemical but with the same or even better efficiency than the best peroxide in performance.

Another parameter to be taken into account at the moment to choose a reagent to treat the polluted soil would be, the time that reagents take to degrade the bulk of contaminants and their stability in the soil. It is very important to know how fast the reactions take in the soil matrix and how long the reagent stay (stability) in the soil, interacting with pollutants. Table 5 gives us an overview of the parameters involved in relation to the time frame of ISCO. From this table can be concluded that hydrogen peroxide acts faster in the bulk of contaminants even though is the less stable.

Table 4. Comparison of different oxidants in terms of cost-effectiveness (Brown, 2003)

Table 5. Comparison of oxidants characteristics, in relation to stability, speed of reaction and maximum T50 of decomposition (Brown, 2003).

By taking the previous information as a base to determine which type of technology and oxidant can be used to treat the soil, it is comprehensible why ISCO technology by applying peroxide-Fenton's reagent is the preferred one. After all, it offers a rapid degradation in time (Yin et al. 1999) and the efficiency in cost-effectiveness including also, the stability makes it the best solution to be taken into account at the moment to choose. But, despite the ample scope in characteristics of the reactions occurring in the subsurface, and the wide variety of reagents used by ISCO, there are some key points related to either sustainability and/or cost-effectiveness, which keep the door opened for an optimization of this technology.

For instance, due to the non-selective characteristic of the reagents (Watts et al. 1994; Karpenko et al. 2009), there would be a loss of oxidants in the subsurface, being as, these would react with the organic matter present in the soil matrix (Upsoil, 2009; Watts et al. 1994, Karpenko et al. 2009, ITRC, 2009), this would increase the money invested in this treatment due to the addition of more oxidant to the soil to finally degrade the hydrocarbon as much as possible. Additionally, ISCO has shown to be more effective at lower pH (2 - 4) (Watts et al., 1996) when a chelator for the catalyst is not present in the treatment (Pignatello et al., 1994, Sutton et al. 2010, Jacobs et al., 2003). This very important since such low pH can considerably diminish the microbial growth rates, likely impinging the diversity of microbes that could be reestablished on the spots where the oxidant was applied (Landa et al., 1994; Sahl et al., 2006).

Biological Remediation

Bioremediation is an ecologically acceptable technology that uses microorganisms to efficiently degrade pollutants such as oil and oil products in the environment (Molina-Barahona et al. 2003). The right microbes are bacteria or fungi, which are capable to degrade contaminants due to their physiological and metabolic capabilities (Boopathy, 2000). During the last decade a lot of microbial cultures were set apart according to the capacity to debase distinct diesel compounds under aerobic, anaerobic, denitrifying, iron-reducing and sulfur-reducing conditions (Evans et al., 1991; Rabus et al., 1993). For instance, a bacteria able to degrade toluene and/or m-xylene under denitrifying conditions, was isolated by Hess and associates in 1997 from a fuel-contaminated aquifer in Menziker - Switzerland.

Tasker, R. in 1988, was also able to isolate three different bacteria (Pseudomonas vesicularis and two different strains of Pseudomonas aeruginosa from a bunker oil-saturated soil sample), capable to use diesel fuel as a sole source of carbon. Table 6 shows the three different streams of bacteria capable to degrade diesel fuel. Up to now, microbial biofilms have shown to be very effective in the reduction of hydrocarbons in the bulk of contaminants at different substrates like activated sludge, aquifers, soils and extreme habitats (Rodriguez-Martinez, 2006).

The application of intrinsic bioremediation or natural attenuation technology as a cleanup system for petroleum contaminated soils and groundwater, has increased during the last decade. Starting in 1995, intrinsic bioremediation has become one of the most important treatments for groundwater decontamination and the second for soil decontamination with hydrocarbons (Rodriguez-Martinez, 2006). This is due to; this technique frequently addresses multiphasic, heterogeneous environments, such as soils in which the contaminant is present in association with soil particles, dissolved in soil liquids, and in the soil atmosphere (Boopathy, 2000).

As it has been stated, this process can reduce organic pollutants such as hydrocarbons, to undetectable concentrations or below the limits established as safe to all the living organisms and the environment (Rodríguez-Martínez, 2006; Terranova Biosystems, 2009). Figures 1 and 2, shows the reliability of bioremediation on treating oil contaminated soil. This technology also takes advantage of natural processes, which can clean a site without having to move them somewhere else; besides, workers will never come into contact with the pollutants in the subsoil.

Table 6. Identification of bacterial isolates obtained from oil contaminated soil

capable of using diesel fuel for growth.

Figure 1. Light and Heavy hydrocarbons compounds degraded by ex-situ bioremediation process (Terranova Biosystems, 2009).

But the main advantage according to sustainability would be that, by using this technique, the release of harmful volatiles compounds that can likely be released during chemical treatments, might not be emitted by this technology. Since microbes change this harmful gases into water and some other less noxious gases like CO2 and a very few waste production, if any is created (EPA, 2001)

In terms of cost-effectiveness, this technology would reduce costs drastically, both in time, resources, money, and fighting negative press, since here the equipment, the labor hours, resources, etc., that might be applied by some other technologies for the soil treatment, would be relatively low (EPA, 2001; Boopathy, 2000; Terranova Biosystems, 2009).

Figure 2. Light and Heavy hydrocarbons compounds degraded by bioremediation process (Terranova Biosystems, 2009).

However, bioremediation has its own limitations which do not leave the microbes to exert all their capacity to degrade some other compounds. For instance heavy metals, radionuclides, and some other chlorinated compounds are not amenable to be biodegradated. Furthermore, some toxic metabolites could also be produced in some cases, due to the microbial metabolism (Boopathy, 2000), which would diminish the colonies to a lower number of individuals. Another issue has to do with the time required by microorganisms to decompose the bulk of contaminants; bioremediation is a relatively slow process which generally takes from several months to several years to complete (Sutton et all., 2010; Jeffries Group, 2010; Tasker, 1988). One main variable affecting the performance of micro-organisms in bioremediation technology has to do with the ability and availability of reduced organic materials, which would work as energy sources in the system. That is, the average oxidation state of carbon in the material, would determine if the pollutant could help the aerobic heterotrophic bacteria, as an effective energy source (Boopathy, 2000). Table 7 summarizes the main factors affecting the bioremediation process.

Table 7. Factors affecting the bioremediation process (Boopathy, 2000).



Growth until critical biomass is reached

Mutation and horizontal gene transfer

Enzyme induction

Enrichment of the capable microbial populations

Production of toxic metabolites


Depletion of preferential substrates

Lack of nutrients

Inhibitory environmental conditions


Too low concentration of contaminants

Chemical structure of contaminants

Toxicity of contaminants

Solubility of contaminants

Biological aerobic vs. anaerobic process

Oxidation/reduction potential

Availability of electron acceptors

Microbial population present in the site

Growth substrate vs. co-metabolism

Type of contaminants


Alternate carbon source present

Microbial interaction (competition, succession, and predation)

Physico-chemical bioavailability of pollutants

Equilibrium sorption

Irreversible sorption

Incorporation into humic matters

Mass transfer limitations

Oxygen diffusion and solubility

Diffusion of nutrients

Solubility/miscibility in/with water

Extreme environmental conditions in the substrate can also affect the process. Conditions such as low temperature, high contents of salt, low pH, low level of nutrients or high concentrations of contaminants can be toxic to micro-organisms (Romantschuk et al., 1999; Sutton et all., 2010; Boopathy, 2000; Jeffries Group, 2010) and they would also reduce the number of individuals. Besides, the spatial distribution of contaminants in relation to degrading organisms and solubility of the same, are well related (bioavailability). Since the rate at which microbial cells can convert contaminants during bioremediation, it is strictly linked to the rate of pollutant uptake, metabolism and the rate at which this contaminant is transferred to the cell (Boopathy, 2000; Romantschuk et al., 1999; Jeffries Group, 2010). Therefore, a higher biotransformation rate would not be the result of an increasing microbial conversion capacity, especially when the mass transfer is a limiting factor (Boopathy and Manning, 1998).

So, you mention bioavailability, toxicity of the contaminant, availability of nutrients, pH, and temperature. What about redox state? This is VERY important.

Fine-tuning the coupling of ISCO and Bioremediation technologies

It is clearly seen that the disadvantages and advantages of one technology are somehow connected to the disadvantages or advantages that the other technology may have. For example, ISCO has shown to be very efficient at a very low pH, but at the same time this low pH would seriously inhibit the startup of bioremediation technology, since at such low pH, bacteria can hardly exist and so on.

That is why, it is intended in this research work to minimize the ups and downs that the two treatments can cause each other (table 8), and help these two technologies reach an optimum level of efficiency where they can work together without interfere one another, with their performance.

Table 8. Performance characteristics of current technologies according to some parameters affecting the coupling; green (+): perceived as scoring good on the dimension indicated; (-): perceived as scoring less well; (?):doubt in the behavior.




Soil Matrix

as a source of nutrients

Addition of Fenton´s









At Once




























By manipulating in the lab parameters like, pH, nutrients and the addition of Fenton-like reagent to the polluted soil, this optimum level of performance would be possible to reach. Previous research showed that, the manipulation of parameters like, Fenton's reagent or processes like redox, exhibited good results for the improving of ISCO technology, at the moment to treat soil polluted with hydrocarbons (Yin et al. 1999). For instance by adding a base to the system, right after Fenton's reagent finishes its job, we could raise the pH to neutral level, in this way we might ensure the startup of bioremediation a bit faster than at normal conditions. Research has been conducted to prove that microorganisms, even after applying in situ chemical oxidation, can re-start after sometime, their decay activity of the contaminants in the soil (Sutton et al. 2010; Chris et al., 2005; Sahl et al., 2007).

According to Goi et al. 2006, by using ISCO as a pretreatment, we can improve bioremediation process, since, the complex pollutants during oxidation reactions will be broken down into a simpler ones, that can be easily degraded by microorganisms and in this way improve the bioavailability of the pollutants in the system, inasmuch as, there will be more parent compounds in the system for microorganisms, and the production of bioavalaible and biodegradable oxidized daughter compounds will be increased as well (Sutton et al. 2010). Moreover, as hydrogen peroxide decomposes the use of ISCO will additionally supply oxygen to the microbial community for improving biological remediation (Goi et al. 2006; Sutton et al. 2010; Sivasubramaniam, 2005; Brown, 1991). Further discussions would be based on how to add the peroxide to the system to increase the efficiency of ISCO in providing bioavailability of the compounds, and the necessary amount of oxygen to keep the system aerobically. Since, by optimizing the oxidant loading (dose concentration and delivery), the effectiveness of the coupling would be maximize and the soil disturbance would be minimize (Upsoil, 2009; Haselow et al., 2003; Mumford et al., 2004; Nelson et al., 2001).

Biostimulation is also an important process occurring in the lab to enhance the biological remediation. The addition of nutrients would exert a pression on microbial growth, increasing at the same time, the biological degradation of pollutants in the system (Zytner at all., 2006). Meaning that the more microorganisms in the system, the more biodegradation. But the characteristics of the soil will determine also the presence or absence of those nutrients in the particles,. By using ISCO the biostimulation process could be achieved in the subsurface, inasmuch as, it is possible to release nutrients through the oxidation of soil organic matter (Sutton et al., 2010; Sirguey et al., 2008; Westersund et al., 2006). Suggesting that, although the addition of nutrients at a proper rate or in excess may improve bioremediation, an alteration in quantity might not be necessary (Sutton et al., 2010). Hence, the aim of this research according to nutrients is well related to the loading rate of peroxidant. Since it is pretended to release nutrients embedded in the soil particles, to favor bioremediation by adding it sequentially. In this way, ISCO could efficiently degrade contaminants chemically and at the same time provide the necessary nutrients so that bacteria can adapt themselves to an aggressive environment and start degrading the simpler compounds, biologically in a short time-frame.

In this way, by controlling at will of the previous parameters, the optimization of the coupling of these two technologies will be guaranteed.

Fine tuning section: some reorganization is required.

First: Discuss the parameters that are improved by coupling ISCO with bioremediation- bioavailability, aerobic conditions, etc.

Second: go deeper into the items that still need to be optimized (and that we will study)- addition of reagent, pH, and nutrients+ matrix

Theoretical Background

A lot of the stuff in these sections (chemical oxidation and bioremediation) was either mentioned above, or should be. And, some of the stuff mentioned above could come here. I would say either combine the sections to make one ISCO section and one bioremediation section, or more clearly split them. We can discuss.

Chemical Oxidation

This process rapidly treats contaminated soils with toxic and recalcitrant organic wastes (Jacobs. 1995, 1996, 1997). It is widely used in both soil and waste water. Due to the widely spectrum of compounds to be treated, and the applicability and aggressiveness of the compounds used by it, the coupling of this process with other delivery technologies, has increased attention (Yin et al. 1999).

In-situ oxidation is based on contact chemistry of the oxidizing agent which reacts with petroleum hydrocarbons, turning them into mineralized products like CO2, salts, and readily biodegradable organic fragments (U.S. Peroxide Company. 2010). Some oxidants are stronger than others (ITRC. 2005). Table 9 lists the relative strengths of the most common oxidants by using chlorine as a reference.

Table 9. Oxidants Strengths (ITRC. 2005)

Many variables like, temperature, pH, concentration of the reactants, catalysts, reactions by-products, and system impurities (e.g., natural organic matter, oxidant scavengers, etc.) need to be considered concurrently to find out the rate of reactions (ITRC 2005). Figure 3 shows three out of the four major factors implied in establishing, if an oxidant will react with some pollutant in the field. The fourth one has to do with the oxidant application technique, being as, it is required that the oxidant must be evenly distributed, throughout the area involved in the treatment. A few out of the more persistent species of contaminants can be consumed by stronger oxidants. The problem is that, these stronger oxidants can be absorbed very fast by the subsurface, avoiding these compounds, to travel along the whole polluted area (ITRC. 2005).

The two most common liquid oxidizers used in soil and groundwater remediation are hydrogen peroxide and potassium permanganate. These two oxidizers are non-selective and will oxidize the contaminants, as well as natural organic material. For instance tree roots, organic carbon, etc. (Jacobs et al. 2003). These oxidants desorb pollutants embedded to the soil particles. An overview of the characteristics of the two common oxidizers is displayed in table 10.

Figure 3. Factors influencing reactions

Table 10. Summary of advantages and limitations of the two common oxidizers ( Jacobs et al., 2003)

Fenton´s chemistry

Hydrogen peroxide is a very well known and very common compound in the commerce; people can buy it at low concentrations as a bactericidal to treat injuries. Pure or in aqueous solutions are clear liquids resembling water, but with a slightly sharp and distinctive odor (Jacobs et al. 2003). Despite its oxidant power, it is not fast enough to degrade many organic contaminants at low concentrations (<0.1%), before decomposition starts (ITRC 2005). For In Situ Chemical Oxidation, it is commonly used together with Fe(2+) to form Fenton's reagent (Yin et al. 1999), in honor to British Professor H.J.H. Fenton (1893-1894). The metal catalysts is regularly supplied by iron oxides inside the soil or added separately as a solubilized iron salt, such us iron sulfate (Jacobs et al. 2003). Moreover, to speed up the chemical reactions and make them more efficient, a low pH (2-4) should be maintained in the system. This adjustment of pH is achieved through the addition of sulfuric acid (H2SO4) to the system. (Yin et al., 1999; ITRC 2005; Jacobs et al., 2003).

When Hydrogen peroxide is pumped into the subsurface at concentrations of 10 to 35%, hydroxyl radicals (OHËš) and water are formed. The previous can be used to rapidly mineralize hydrocarbon and some other contaminants to water and carbon dioxide (CO2) (Jacobs et al. 2003). This reaction is enhanced in the presence of iron, equation 1.

Equation 1: Fe2+ + H2O2 à OHËš + OH- + Fe3+

If pH is less than 5, iron(III)(Fe3+) can be reconverted into iron (II)(Fe2+) again, keeping this in solution to continue the production of hydroxyl radicals, (ITRC, 2005). Equation 2 shows how a subsequent reaction with another hydrogen peroxide molecule, the iron(II)(Fe2+) is regenerated (Metelitsa, 1991).

Equation 2: Fe3+ + H2O2 à Fe2+ + HO2Ëš + H+

The hydroxyl radical is non-selective radical which allows it to attack any C-H bonds present in any organic molecules, making it a very strong degrader of many solvents, haloalkanes, esters, aromatics, and pesticides (Haag and Yao, 1992). Table 11 shows the level at which Fenton's reagent act on them, in comparison with some other oxidants.

Table 11. Level at which Fenton's reagent act in several compounds (Brown, 2003)


The main goal of microorganisms degrading petroleum hydrocarbons during bioremediation process is to take the chemical contaminants in the subsurface and by way of reactions like oxidation-reduction, metabolize the desired pollutant and turn it into useable energy source to favor the microbial growth. Here the metabolites are usually less toxic than the original ones, being as, carbon dioxide and water are formed at the end of this process (Nester et al., 2001; Boopathy, 2000; Donlon et al., 2010). An important requirement to achieve an optimal biodegradation rate during the process is the presence of microorganisms with the proper metabolic capacities (Das et al., 2010).

Microbial attack to hydrocarbons can be ranked according to the susceptibility of the hydrocarbons during the process. Generally, the normal process start by tackling the linear alkanes, followed by the branched alkanes, small aromatics and finally the cyclic alkanes (Ulrici, 2000; Perry, 1984), some other compounds present in petroleum hydrocarbons are not degraded at all, this the case for PAHs (Atlas et al., 2009). As stated previously in this document, the microorganisms capable to degrade hydrocarbons are bacteria, yeast, and fungi. The efficiency for soil bacteria as they are the most active agents and also the first microorganisms on removing pollutants off the soil during the biological process (Rahman et al., 2003; Brooijmans et al., 2009), has been ranged from 6% (Jones et al., 1970) to 82% (Pinholt et al., 1979). It is suggested by many scientists that, to degrade complex mixtures of hydrocarbons like crude oil in soil, might be used mixed populations of microorganisms with an overall enzymatic capacity, at a proper temperature, where the maximum rate of degradation can be reached (Fig 4) (Bartha et al., 1984).

Fig 4. Hydrocarbon degradation rates in soil, fresh water and marine environments (Bartha et al., 1984).

Mechanisms and compounds involved in degradation of petroleum hydrocarbons.

The highest efficiency of biological degradation is brought about during aerobic conditions, such conditions, metabolize the hydrocarbons by including oxygen as a reactant during the process (Boopathy, 2000; Fristche et al., 2000), the hydrocarbon loses electrons and is oxidized while oxygen is reduced by gaining those electrons (Donlon et al., 2010). Fig 5 shows the way how microorganisms make the aerobic degradation of hydrocarbons (Fristche et al., 2000). This reactant inclusion (oxidative process), is the primary intracellular attack to pollutants performed by organic compounds (enzymes) like oxygenases and peroxidases, where the activation and addition of oxygen, is the main reaction reaction catalyzed by them (Das, 2010). Then, the pollutants are little by little converted into intermediates of the central intermediary metabolism, through the peripheral degradation pathways, for instance, the tricarboxilic acid cycle (Das, 2010). Processes like biosynthesis of cell biomass, are produced as from the central precursor metabolites, an example would be acetyl-CoA, succinate and the pyruvate. Compounds like sugar which are necessary for growth and work in some other biosynthesis process, are synthesized by gluconeogenesis. (Das, 2010). Fixations of microbial cells to substrates and biosurfactants formation are other mechanisms employed by the microorganism to degrade the hydrocarbons (Hommel, 1990). Fig 6 shows the primary intracellular attack to organic pollutants, deployed by oxygenases (Fristche et al., 2000).

Fig 5. Principle for aerobic degradation of hydrocarbons by microorganisms (Fristche et al., 2000).

Heme-thiolate Monooxygenases play a main role in microbial degradation of petroleum hydrocarbons; one of the main group of enzymes constituting these monooxygenases is the Cytocrhome P450 alkane hydroxylases (Van Beilen et al. 2007), which at the same time are contained into eukaryotes cells in several P450 forms (Table 12). Their role is to contribute as a join of isoforms to the metabolic conversion of a given substrate, by introducing oxygen for the beginning of biodegradation process (Figure 6). This Multiplicity can only be found in some species of these P450 (Zimmer et al., 1996). In reality, the ability to use n-alkanes and aliphatic hydrocarbons as a unique source of energy and carbon for a varied species of yeast, is due to the existence of multiple forms of microsomal cytochrome P450 (Scheuer et al., 1998).

Another mechanism employed by microorganisms in the biological degradation of hydrocarbons is the production of biosurfactants. A miscellaneous group of surface active chemical compounds (Muthusamy et al., 2008; Mahmound et al., 2008; Ilori et al., 2005; Ilori et al., 2008; Obayori et al., 2009), that enhance solubilization and removal of contaminants (Brusseau et al., 1995; Bai et al., 1997) as well as biodegradation due to the increasing of bioavailability of contaminants (Barkay et al., 1999). A research conducted by Cameotra and Singh in 2008, determined an efficiency of 90% on removal of hydrocarbons from a soil contaminated with oily sludge, in 6 weeks by using a consortium of microorganisms (Pseudomonas aeruginosa and an isolate Rhodococcus erythropolis) in liquid culture.

Table 12. Summary of enzymes involved in biodegradation of petroleum hydrocarbons (Das et al 2010).

During the research, it was evaluated the capacity of the consortium to decompose the sludge hydrocarbons and the effect of the two additives (a combination of nutrients and the elaboration of a natural biosurfactant) by separate and altogether, on the effectiveness of the process. The biosurfactant applied was identified as being a mixture of 11 rhamnolipid congeners and it was produced by a consortium member. The overall efficiency of hydrocarbon degradation on a soil contaminated with 1% (v/v) crude oil for the consortium was 91%, in 5 weeks. Separate application of any additive together with the consortium, emitted an efficiency of 91 - 95% in 4 weeks. But the best performance of the treatment (98%) was obtained when the two additives were added along with the consortium, providing the data enough to consider the use of a natural biosurfactant for biological hydrocarbon degradation (Das 2010). Table 13 is a summary of fresh reports about biosurfactant production by different microorganisms. Pseudomonas is the best known bacteria with the capacity to use hydrocarbons as a source of energy and produce biosurfactants (Cameotra et al., 2008; Pornsunthorntawee et al., 2008).

Biosurfactants can also work as emulsifying agents, since they can reduce the surface tension and create micelles. The microdroplets enclosed in a capsule in the hydrophobic microbial cell surface are taken inside and decomposed (Das et al., 2010). Figure 7 shows the involvement of biosurfactant (rhamnolipids) elaborated by Pseudomonas sp. and how the micelles are made in the uptake of hydrocarbons (Fristche et al., 2000).

Fig 6. Enzymatic reactions involved in degradation of petroleum hydrocarbons (Fristche et al., 2000).

Table 13 List of biosurfactants produced by microorganisms (Das et al., 2010)

Figure 7. Involvement of biosurfactant (rhamnolipid) produced by Pseudomonas sp. in the uptake of hydrocarbons (Fristche et al., 2000).

Bioremediation section: this is a very technical description of the biochemistry of microbial hydrocarbon degradation. Its nice to understand what occurs on a microbial level, but this has nothing to do with what we will be doing in the lab. We will be optimizing conditions for bioremediation. Thus, it is much more important to describe previous in situ bioremediation projects, what was done in these projects, what has been learned about the technology, and how it still needs to be improved.

Research Objective

To optimize the coupling of In Situ Chemical Oxidation (ISCO) and Bioremediation technologies, by manipulating parameters like pH, Nutrient contents and Oxidant's addition.

General Research Question

What are the minimum levels that parameters involved in ISCO process must achieve, to allow Bioremediation start in the shortest time frame?

Research and Sub-Research Questions

What is the impact of pH in the efficiency of the coupling of ISCO and Bioremediation technology?

Does the addition of a base to raise the pH from low to neutral, improve the efficiency of soil decontamination made by the coupling of ISCO and Bioremediation technology by applying old Fenton's at pH 2 - 4?

Can the low efficiency of ISCO technology at neutral pH by applying modified Fenton's for soil decontamination be leveled by Bioremediation process at the same pH?

What is the impact of timing the addition of peroxide to the soil, in the bulk contaminant degradation and startup of Bioremediation process?

Can the efficiency in hydrocarbon degradation made by the coupling of ISCO and Bioremediation technologies, be improved by adding the total amount of peroxide at one time to the system?

Can the efficiency of hydrocarbon degradation during ISCO process be improved by adding sequentially the peroxide when ISCO and Bioremediation technology are coupled?

What is the impact of soil type in relation to ISCO performance and biostimulation through nutrients and the startup of Bioremediation process?

Can the degradation of the bulk of contaminants in the coupling of ISCO and Bioremediation technology be improved at sandy, peat and clay soil by improving nutrients release due to a regulation in the load of the Fenton's reagent during the ISCO process?

Materials and Methods

Sandy, clay and peat soil samples with a high saturation of petroleum-hydrocarbon pollutant were collected at different sites in the railway station in Wegliniec (Polland).

The laboratory analysis will be conducted into three setups:

The first setup will be carried out to analyze the efficiency in the addition of Fenton`s reagent sequentially or at one time, for this analysis a total number of 48 serum bottles will be used: 12 for ISCO sequentially added and 12 for ISCO added at one time, the other 24 bottles will be kept for control of the treatment, 12 bottles for no ISCO control and 12 for Biological control.

The second setup has to do with pH; a same number of bottles will be used to measure the efficiency of the coupling at low pH (12 serum bottles) and at normal pH (12 serum bottles). Same number of serum bottles will be applied for control of the treatment, 12 bottles for ISCO control and other 12 for bioremediation control.

The third setup will be applied for the analysis of nutrient releasing due to the addition of ISCO to the three types of soil, sandy, clay and peat. Because of the no clear protocols to measure nutrients in the samples, the number of bottles to be used, or the exact setup for measuring nutrients, cannot be determined yet.

For the previous setups except for nutrients, an amount of 4 grams of soil will be put into the serum bottles and a certain amount of Fenton's reagent. The amount as well as the concentration for this Fenton´s reagent will be discussed later with the supervisor. The content of the bottle will be mixed a little, so that the reactions can start. After that, the bottles with no lid will be taken to a shaker at a temperature of 20 °C or 30 °C where they will start mixing really well with the oxygen and then it will proceed to measure. The bottles will not have any lid or stopper to make sure that oxygen penetrates the samples and mix really well with the content, in this way we also assure the treatment will be carried out under aerobic conditions. If there is any use of stoppers for the bottles, mechanisms for the insertion of oxygen or flushers will be necessary to provide, so that the treatment can be accomplished in a correct way.

From time to time, a number of bottles per setup will be sacrificed to proceed to make the TPH extraction and measure the progress of the treatment; these measurements will be effectuated in a Gas Chromatographer machine, specially calibrated for this process.

What about expected results section?

Juan Pablo,

Its looking very good. For the last proposal, I stated that you need to describe more clearly why a parameter is important, and you have improved that quite well. The research questions are also coming along well. There are some organizational problems still, but we can discuss that later. Also, I would like to see the "fine tuning of the coupled technology" section to be expanded and improved. Moving this section to just before your research questions would provide a good introduction to the research questions that we will address.