**4. Mineralization of NAPL**

The most widely applied soil bioremediation approach to organic contaminants involved the biostimulation of natural microbial biodegraders. Biodegradation requires a source of carbon (organic contaminant) and nutrients, as amendment. The hydrophobic organic contaminants represent the carbon source as electron donors, while nitrogen and phosphorous are essential for microbial growth for cellular metabolism. Addition of nitrogen particularly is often necessary due to heavy demands by the biodegradation process. Phosphorous is usually amended in lower concentration. Optimizing nutrient status of a contaminated soil can have direct impact on microbial activity and contaminants biodegradation. In some instances, the negative effects of high nutrients amendment with NPK on soil biodegradation especially on aromatics have been reported [19–21].

The ultimate microbial aerobic degradation process of converting bioavailable NAPL constituents in a contaminated soil matrix:

This process is commonly referred to as mineralization. The degradation process is brought about under aerobic conditions. NAPL constituents are hydrophobic organic chemicals that exhibit limited or no solubility in contaminated soils and thermodynamically tend to partition to the soil solid phase. Sorption may account for more than 95% of the total contaminant mass. As a consequence, the hydrophobic contaminant exhibits limited dissolved mass transfer phase and bioavailability, which limits its biotic degradation in the soil. Therefore, in a contaminated soil environment, biodegradation of an organic hydrophobic compound should be envisioned as a stepwise process involving contaminants bioavailability and species of biodegraders.

The use of surfactants represents a cost-effective and promising method that can enhance bioremediation of organic hydrophobic contaminants in soils. Many studies have shown that surfactants can solubilize and mobilize hydrophobic organic contaminants sorbed onto soil matrices [22–24]. Adding surfactant to a contaminated soil matrix is expected to enhance microbial degradation through mobilization or emulsification. Mobilization takes place at concentrations below CMC and the solubilization process above the surfactant CMC, whereas emulsification allows for dispersion of one phase into the other. A certain amount of surfactant in the slurry system will inevitably be sorbed onto the soil particles. Sorbed surfactant does not contribute to the solubilization and bioavailability of contaminants during treatment. The more surfactant is sorbed, the less effective will be the surfactant. Furthermore, soil hydrophobicity may increase as more surfactant becomes sorbed onto the contaminated soil matrix.


**Table 4.** Relevant environmental and surfactant considerations for *ex situ* surfactant-enhanced bioremediation.

Surfactants can enhance metabolic degradation and thereby, contaminants mineralization in the soil by two main mechanisms [25]. One mechanism involves the increase in the contaminant bioavailability for microorganisms. The second mechanism is due to interaction with cell surface resulting in the hydrophobicity increases in the cell surface allowing hydrophobic organic chemicals to interact with bacterial cells. Environmental factors and surfactant properties affecting the metabolic capability of biodegraders in the soil vis-à-vis hydrophobic organic contaminants are summarized in **Table 4**.

The role of treatability studies for *ex situ* surfactant-enhanced bioremediation of hydrophobic organic contaminants contaminated soil is vital. It will allow to derive crucial information that will serve as blueprint to optimize field operation. Typically, a treatability study will be conducted in laboratory microcosms to inform (a) on the dosage of surfactant required to optimize contaminant mass transfer, (b) on the effect of temperature on contaminant bioavailability as temperature may affect surfactant efficiency and microbial activity, (c) on optimum biostimulation through the addition of appropriate nutrient amendments such as N, P and other elements, (d) optimum moisture level as it will vary with soil type, (e) selection of appropriate surfactant, (f) modeling rate of contaminants degradation under varying environmental factors, (g) rate of oxygen and nutrients consumption under different environmental conditions, (h) implement bioaugmentation utilization by inoculation with acclimated bacteria strains, (i) the complimentary effects of combined bioaugmentation and biostimulation, (j) determine whether targeted level of cleanup is attainable, (k) formulation of an efficient and effective monitoring program for field treatment operation, (l) the engineering design, (m) potential surfactant toxicity and means to reduce it, (n) sorption behavior of a surfactant.

The two main strategies can be highlighted for assessing a bioremediation system performance. A material balance approach consists of extracting and quantifying residual parent compounds and monitoring partitioning in the headspace phase. The other strategy involves monitoring the system for CO2 production. A direct correlation occurs between mineralization of the parent compound and CO2 production.

The biodegradation during the treatability assessment may be modeled through either a firstor zero-order power rate model [26]. A zero-order reaction indicates the biodegradation of a parent contaminant in the microcosm occurs at a constant rate and independent of concentration and time. If the parent compound C is mineralized to CO2, the rate of disappearance of C is given by Eq. (9):

$$\mathbf{d} \mathbf{C} / \mathbf{d} \mathbf{t} = -\mathbf{k} \tag{9}$$

integration yields Eq. (10):

$$\mathbf{C}\_{\text{t}} = \mathbf{C}\_{\text{o}} - \mathbf{k}\mathbf{t} \tag{10}$$

where

to the soil solid phase. Sorption may account for more than 95% of the total contaminant mass. As a consequence, the hydrophobic contaminant exhibits limited dissolved mass transfer phase and bioavailability, which limits its biotic degradation in the soil. Therefore, in a contaminated soil environment, biodegradation of an organic hydrophobic compound should be envisioned as a stepwise process involving contaminants bioavailability and species of

The use of surfactants represents a cost-effective and promising method that can enhance bioremediation of organic hydrophobic contaminants in soils. Many studies have shown that surfactants can solubilize and mobilize hydrophobic organic contaminants sorbed onto soil matrices [22–24]. Adding surfactant to a contaminated soil matrix is expected to enhance microbial degradation through mobilization or emulsification. Mobilization takes place at concentrations below CMC and the solubilization process above the surfactant CMC, whereas emulsification allows for dispersion of one phase into the other. A certain amount of surfactant in the slurry system will inevitably be sorbed onto the soil particles. Sorbed surfactant does not contribute to the solubilization and bioavailability of contaminants during treatment. The more surfactant is sorbed, the less effective will be the surfactant. Furthermore, soil hydrophobicity may increase as more surfactant becomes sorbed onto the contaminated soil matrix.

Acclimation Proper biodegraders; enzymatic adjustment for metabolic process

Nutrients Sufficient N, P not limiting biodegraders growth; C:N:P ratio of 100:50:1

Effective concentration Efficient in increasing aqueous solubility of organic compounds at

CMC Effective below CMC; partial micelle encapsulation of contaminant; low sequestration vis-à-vis target contaminant

**Table 4.** Relevant environmental and surfactant considerations for *ex situ* surfactant-enhanced bioremediation.

System slurry Optimized to promote mass transfer; 50–80% of soil water intrinsic saturation

biodegraders.

**Considerations Remarks**

Metabolites Non-toxic

Temperature Mesophiles 15–45°C Oxygen Aerobes; DO > 0.30 mg/L pH Optimum range 5–9

Redox potential Greater than 70 mV; promote aerobes

318 Soil Contamination - Current Consequences and Further Solutions

Salinity Low inhibition of CMC formation

Environmental risk Pose no risk to the environment

Substrate source Not a preferential growth substrate Sorption behavior Low sorption onto soil constituents

Toxicity No inhibitory effects; not toxic to any receptors

low concentration Recalcitrancy Non-persistent; biodegradable and mineralizable

*Environmental factors*

*Surfactant properties*

Ct = parent compound present at time t

Co = initial concentration of parent compound

k = zero-order reaction rate constant

t = corresponding sampling time.

First-order reactions have rates that depend on mass transfer of parent compound concurrent to its biodegradation, Eq. (11):

$$\text{dC/dt} = -\text{kC} \tag{11}$$

where

C = parent compound concentration

t = corresponding sampling time

k = first-order reaction rate constant integration yields Eq. (12):

$$\text{Ln}\left(\text{C}\_{\text{t}}\right) - \text{Ln}\left(\text{C}\_{\text{o}}\right) = \text{Ln}\left(\text{C}\_{\text{t}}/\text{C}\_{\text{o}}\right) = -\text{kt} \tag{12}$$

where

Ct = parent compound present at time t

Co = initial concentration of parent compound

k = first-order reaction rate constant (time−1)

t = corresponding sampling time.

Solving for concentration yields Eq. (13):

$$\mathbf{C} = \mathbf{C}\_o \mathbf{e}^{-\text{kt}} \tag{13}$$

and parameters are as defined above.

Biosurfactants may be the strategic choice for increasing contaminant bioavailability in bioreactors while minimizing toxicity to biodegraders. An examination of the literature indicates that synthetic surfactants while effective for increasing contaminant mass transfers at the recommended concentration may show inhibitorial effects on the microorganisms in the bioreactor [27, 28]. In such case, this will inhibit cell proliferation and thus the biodegradation of organic contaminants. According to empirical evidence, surfactant toxicity was found to be primarily dependent on its molecular structure, in order of toxicity, generally non-ionic <anionic < cationic [28]. Several practical approaches may be implemented to reduce surfactant cytotoxicity in a bioreactor by considering a suitable biosurfactant as an alternative to a synthetic surfactant, adding a surfactant at concentration below CMC, using a suitable nonionic surfactants, using a suitable combination of biosurfactant and synthetic surfactant, in some instances, strategically increasing the surfactant concentration to decrease contact of biodegraders with the contaminant, prescreening for a suitable additive such as Ca and Mg as they were found to stabilize the cell membrane, thereby decreasing surfactant toxicity [29].
