**3. Common substrates and additives used for bioremediation**

To stimulate microbial growth and degradation of contaminants, supplemental amendments including those that directly support microbiological growth (C, N, P) and those that maintain or create favorable geochemistry (pH buffering, dissolved O2) are used. In some cases, surfactants are also used to enhance solubility and bioavailability of contaminants from soil and sediments in order to improve treatment efficiency [19-21].

#### **3.1. Organic carbon substrates**

As the major building block for microorganisms, organic carbon may be the most important and prominent additive used in bioremediation. Under anaerobic conditions, many microor‐ ganisms are capable of fermentation of organic matter, and some bacteria can produce hydrogen gas. Thus, almost any fermentable substrate can be a potential source of carbon and hydrogen to stimulate bioremediation. These include naturally occurring dissolved organic carbon (DOC), accidental releases of anthropogenic carbon (e.g., fuels), carbohydrates (sugars), alcohols, oils, solids (e.g., bark mulch, chitin), and complex compounds (e.g., whey and cellulose) [22-23]. Table 1 summarizes the attributes of several common substrate types. These substrates are generally classified into three types (soluble, slow release, and solid substrates), and each type will be discussed in more detail in the following sections.


**Table 1.** Substrates used in bioremediation (modified from [24-25])

#### *3.1.1. Soluble carbon substrates*

**3.1. Organic carbon substrates**

148 Advances in Bioremediation of Wastewater and Polluted Soil

As the major building block for microorganisms, organic carbon may be the most important and prominent additive used in bioremediation. Under anaerobic conditions, many microor‐ ganisms are capable of fermentation of organic matter, and some bacteria can produce hydrogen gas. Thus, almost any fermentable substrate can be a potential source of carbon and hydrogen to stimulate bioremediation. These include naturally occurring dissolved organic carbon (DOC), accidental releases of anthropogenic carbon (e.g., fuels), carbohydrates (sugars), alcohols, oils, solids (e.g., bark mulch, chitin), and complex compounds (e.g., whey and cellulose) [22-23]. Table 1 summarizes the attributes of several common substrate types. These substrates are generally classified into three types (soluble, slow release, and solid substrates),

*Soluble substrate*

*Slow release substrates*

*Solid substrates*

**techniques Form of application Frequency of application**

recirculation systems Dilute acids or salts in water Continuous to monthly

Injection wells Dissolved in water Continuous to monthly

wells Dissolved in water or slurry Monthly to annually

Straight oil injection with water push or high oil/water content (>20%) emulsions

Low oil/water content (>10%) microemulsions suspended in water

Dilute in water Continuous to monthly

surface amendments One-time application

chitin slurry Solid or slurry Annually or biennially, potential

Annually to biennially for HRC®, every 3-4 years for HRC-X®, potential for one-time application

Typically one-time application

Typically every 2-3 years

for one-time application

and each type will be discussed in more detail in the following sections.

Injection wells or recirculation systems

HRC® or HRC-X® Direct injection Straight injection

wells

wells

Mulch and compost Trenching or excavation Trenches, excavations, or

**Substrate Typical delivery**

Lactate and butyrate Injection wells or

Whey Direct injection or injection

Vegetable oils Direct injection or injection

Vegetable oil emulsions Direct injection or injection

Chitin (solid) Trenching or injection of a

**Table 1.** Substrates used in bioremediation (modified from [24-25])

Methanol, ethanol, sodium benzoate

Molasses, high fructose corn syrup

As shown in Table 1, sodium lactate, molasses, ethanol, methanol, butyrate, and sodium benzoate have been used as soluble substrates, and sodium lactate and molasses are among the most widely used in bioremediation. Soluble substrates are applicable to most site conditions with the exception of aquifers with very high (> 30 cm per day) or very low (<30 cm per year) groundwater velocities. Soluble substrates applied as dissolved or "aqueous" phase offer the greatest potential for uniform distribution throughout the aquifer matrix relative to other substrates. Soluble substrates are easy to handle, mix, and inject. Advection helps soluble substrate distribution in the subsurface. As a result, it is possible to increase the radius of influence (ROI) and reduce the number of injection points, as a larger volume of substrate can be dispersed from a single injection point. Soluble substrates are best suited for remediation of deep aquifers where drilling costs are high.

The following disadvantages associated with the use of soluble substrates need to be recog‐ nized:


#### *3.1.2. Slow-release substrates*

The common slow-release carbon substrates used to stimulate anaerobic bioremediation include HRC® (Hydrogen release compounds) and vegetable (edible) oils. These substrates are intended to be long-lasting in their ability to supply carbon for microbial growth. They are relatively immobile in the subsurface, and rely on advection and dispersion of soluble compounds from the slow-release substrates (e.g., lactic acid for HRC®) for effective delivery throughout the aquifer matrix.

The primary benefit of slow-release substrates is that they require infrequent injection (often only once) with no O&M requirements other than performance monitoring; however, uneven distribution may be an issue for slow-release substrates because of the viscous characteristics of these fluid substrates.

To improve the distribution of slow-release substrates in the subsurface, while still providing a long-lasting source of organic carbon, vegetable oil emulsions have been developed. Microemulsions consisting of 5% to 10% vegetable oil in water (by volume) are relatively lowviscosity mixtures. The use of microemulsions is the result of lessons learned in early vegetable oil field trials. In earlier tests using coarse viscous emulsions or neat vegetable oil, high injection back pressures limited ROI, and reductions in hydraulic conductivity were observed [24-25].

#### *3.1.3. Solid substrates*

Solid substrates that have been used in bioremediation include tree mulch, compost, as well as other agricultural byproducts such as cottonseed hulls. Mulch used in bioremediation is usually obtained from shredding and chipping tree and shrub trimmings. To provide a source of nitrogen for microbial growth and also provide a source of more readily degradable organic carbon, green plant material or compost is often incorporated into solid substrates in these applications. Degradation of the solid substrates by microbial processes in the subsurface provides a number of breakdown products (e.g., humic acids). Solid substrates are intended to be long-term sources of organic carbon, with anticipated lifespans exceeding 5 to 10 years [26-27]. The drawback with the solid substrates also lies in the fact that it is hard to be degraded and used by the microbes as readily as the soluble substrates.

#### **3.2. Other additives used in bioremediation**

#### *3.2.1. Oxygen gas*

In aerobic reactions, microorganisms extract energy via electron transfer during oxidation of contaminants and reduction of oxygen gas. Electrons are removed from contaminants and transferred to oxygen during the process. The major kinetic limitation on aerobic bioremedia‐ tion is often the availability of molecular oxygen due to low solubility of oxygen gas in water. In the absence of any external supply of oxygen, concentration of dissolved oxygen in water quickly decreases to very low levels, resulting in anoxic conditions and disruption of aerobic metabolism.

To promote aerobic biodegradation, air, oxygen, or other oxygen sources (e.g., hydrogen peroxide, ozone, sodium nitrate, and perchlorate) may need to be added in some systems. Depending on their physical properties, site hydrogeology, and the desired delivery efficiency, oxygen and oxygen-releasing compounds can be delivered to groundwater via different methods. There are two methods to introduce oxygen to aquifers: one is direct supply of air into groundwater through aeration wells; the other is through addition of hydrogen peroxide. Dissolved oxygen is released from hydrogen peroxide as the hydrogen peroxide rapidly degrades into water and oxygen gas through hydrolysis [28].

#### *3.2.2. Nutrients*

compounds from the slow-release substrates (e.g., lactic acid for HRC®) for effective delivery

The primary benefit of slow-release substrates is that they require infrequent injection (often only once) with no O&M requirements other than performance monitoring; however, uneven distribution may be an issue for slow-release substrates because of the viscous characteristics

To improve the distribution of slow-release substrates in the subsurface, while still providing a long-lasting source of organic carbon, vegetable oil emulsions have been developed. Microemulsions consisting of 5% to 10% vegetable oil in water (by volume) are relatively lowviscosity mixtures. The use of microemulsions is the result of lessons learned in early vegetable oil field trials. In earlier tests using coarse viscous emulsions or neat vegetable oil, high injection back pressures limited ROI, and reductions in hydraulic conductivity were observed [24-25].

Solid substrates that have been used in bioremediation include tree mulch, compost, as well as other agricultural byproducts such as cottonseed hulls. Mulch used in bioremediation is usually obtained from shredding and chipping tree and shrub trimmings. To provide a source of nitrogen for microbial growth and also provide a source of more readily degradable organic carbon, green plant material or compost is often incorporated into solid substrates in these applications. Degradation of the solid substrates by microbial processes in the subsurface provides a number of breakdown products (e.g., humic acids). Solid substrates are intended to be long-term sources of organic carbon, with anticipated lifespans exceeding 5 to 10 years [26-27]. The drawback with the solid substrates also lies in the fact that it is hard to be degraded

In aerobic reactions, microorganisms extract energy via electron transfer during oxidation of contaminants and reduction of oxygen gas. Electrons are removed from contaminants and transferred to oxygen during the process. The major kinetic limitation on aerobic bioremedia‐ tion is often the availability of molecular oxygen due to low solubility of oxygen gas in water. In the absence of any external supply of oxygen, concentration of dissolved oxygen in water quickly decreases to very low levels, resulting in anoxic conditions and disruption of aerobic

To promote aerobic biodegradation, air, oxygen, or other oxygen sources (e.g., hydrogen peroxide, ozone, sodium nitrate, and perchlorate) may need to be added in some systems. Depending on their physical properties, site hydrogeology, and the desired delivery efficiency, oxygen and oxygen-releasing compounds can be delivered to groundwater via different methods. There are two methods to introduce oxygen to aquifers: one is direct supply of air into groundwater through aeration wells; the other is through addition of hydrogen peroxide.

and used by the microbes as readily as the soluble substrates.

**3.2. Other additives used in bioremediation**

throughout the aquifer matrix.

150 Advances in Bioremediation of Wastewater and Polluted Soil

of these fluid substrates.

*3.1.3. Solid substrates*

*3.2.1. Oxygen gas*

metabolism.

An aquifer normally contains sufficient amounts of nutrients for microbial growth. In engi‐ neered bioremediation, however, due to the addition of organic substrate, the nutritional demand imposed by rapid microbial growth may exceed the capacity of the aquifer system [29]. In addition to a readily degradable carbon source, microorganisms also require nutrients such as nitrogen, phosphorous, and potassium (N, P, and K) for cellular metabolism and therefore successful growth [28, 30].

Commonly used nutrients include mineral salts (e.g., KNO3, NaNO3, Ca(NO3)2, NH4NO3, (NH4)2SO4, K2HPO4, (NH4)2HPO4, MgNH4PO4,), anhydrous ammonia (NH3), urea (NH2)2CO, and many commercial inorganic fertilizers [8]. In practice, nitrogen and phosphorus require‐ ments are often estimated by calculating a carbon to nitrogen to phosphorus ratio C/N/P close to 100/(10 to 5)/1. Many authors report optimum experimental results with a C/N/P of ~70/3/0.6 [31], 8/1/0.07 [32] for crude oil bioremediation.

## *3.2.3. pH modifiers*

The pH range within which bioremediation processes operate most efficiently is approxi‐ mately 5.5 to 8 [8], as this is also the optimal pH range for many heterotrophic bacteria, the major microorganisms active in most bioremediation technologies; however, the optimal pH range for a particular situation is site-specific.

At a field site, pH is influenced by a complex relationship between organisms, contaminant chemistry, and physical and chemical properties of the local subsurface environment. For example, in low-alkalinity systems, fermentation of complex substrates generates acids, and hydrochloric acid (HCl) is formed during anaerobic dechlorination. These processes may significantly decrease groundwater pH. Reducing groundwater pH to below 5 will likely inhibit microbial growth (e.g., sulfate reducers, methanogens, and some dechlorinating microbes) [33]. Normally, the natural buffering capacity of the aquifer matrix is adequate to prevent the development of acidic groundwater pH; however, at some sites, pH buffer amendments such as sodium bicarbonate may be required to maintain near-neutral pH in groundwater systems with insufficient natural buffering capacity. The maintenance of nearneutral groundwater pH is not only important for microbial growth, but also for secondary groundwater geochemistry.
