**4. Additives implementation in bioremediation**

#### **4.1. Organic carbon substrate selection**

The choice of electron donor (substrate) and the delivery methods are essential components of bioremediation. Substrates differ in rates at which they are degraded and become available for biodegradation. They also differ in the complexity of their composition, in their physical form, and in their cost [24, 34].

The selected organic substrate should be suitable for the biogeochemical and hydrodynamic characteristics of the aquifer to be treated. Selection of an appropriate substrate should take into account expected performance in developing appropriate anaerobic reactive zones, the rate at which the substrate is used (efficiency of use), site infrastructure or land use, substrate availability, application configuration, delivery and distribution requirements, system O&M requirements, and cost of implementation (life-cycle cost, including cost of O&M).

The most commonly added substrates in bioremediation include lactate, molasses, hydrogen release compound (HRC®), and vegetable oils. Ethanol, methanol, benzoate, butyrate, highfructose corn syrup (HFCS), whey, mulch, compost, chitin, and gaseous hydrogen are less frequently used. The physical and chemical characteristics of a substrate (e.g., phase and solubility) may make certain substrates more suitable than others in a particular application. Sometimes a single substrate is not sufficient, and in fact, combinations of various substrates are becoming more common in contaminated site remediation. For example, an easily distributed and rapidly degraded soluble substrate, such as lactate, may be combined with a slow-release substrate, such as vegetable oil, as it provides longer term supply of organic carbon. In aquifer systems that are naturally aerobic, an easily distributed and highly degrad‐ able soluble substrate (e.g., ethanol or lactate) can be used to rapidly induce anaerobic, reducing conditions, thus reduce lag phase of anaerobic bacteria. Then a longer lasting, "slowrelease" substrate (e.g., vegetable oil, chitin, or whey) is used to sustain the reaction zone. The combination use of these two types of substrates will minimize the cost of maintaining the treatment system [23-24].

In practice, however, selection of a substrate is often based on contractor experience or familiarity, or as a result of commercial marketing. Substrates are usually selected from a wide variety of available low cost food-grade products such as molasses, vegetable oils, and whey. The potential pitfall of this strategy lies in that ineffective, even inappropriate substrate, may have been selected in some cases, leading to poor treatment results.

#### **4.2. Organic carbon dosage**

The underlying principle in determining substrate loading rate is maintaining the balance in a remediation system so that native electron acceptors are fully utilized, while at the same time sufficient electron donor is left in the system to degrade the contaminant mass. To calculate substrate mass required to deplete available electron acceptors flux, substrate composition, stoichiometry, and utilization efficiency of the anticipated degradation reactions need to be known. However, the exact stoichiometric reactions and electron acceptor flux that occur in a site is difficult, if not impractical, to determine [34]. Calculations for substrate demand can be obtained from theoretical hydrogen equivalents resulted from a known mass of substrate versus estimated electron acceptor demand at a site. The electron acceptor demand typically comprises the following three components:

**1.** Contaminant electron acceptor demand. During anaerobic dechlorination, the contami‐ nant is the electron acceptor, and there is a stoichiometric relationship between electron donor (e.g., hydrogen) and electron acceptor (contaminant mass). The stoichiometric relationships for direct anaerobic dechlorination of CAHs are favorable. For example, on a mass basis, 1 mg of H2 will dechlorinate PCE (21 mg), TCE (22 mg), DCE (24 mg), and VC (31 mg), assuming 100% use of H2 by the dechlorinating microorganisms [25, 35].

for biodegradation. They also differ in the complexity of their composition, in their physical

The selected organic substrate should be suitable for the biogeochemical and hydrodynamic characteristics of the aquifer to be treated. Selection of an appropriate substrate should take into account expected performance in developing appropriate anaerobic reactive zones, the rate at which the substrate is used (efficiency of use), site infrastructure or land use, substrate availability, application configuration, delivery and distribution requirements, system O&M

The most commonly added substrates in bioremediation include lactate, molasses, hydrogen release compound (HRC®), and vegetable oils. Ethanol, methanol, benzoate, butyrate, highfructose corn syrup (HFCS), whey, mulch, compost, chitin, and gaseous hydrogen are less frequently used. The physical and chemical characteristics of a substrate (e.g., phase and solubility) may make certain substrates more suitable than others in a particular application. Sometimes a single substrate is not sufficient, and in fact, combinations of various substrates are becoming more common in contaminated site remediation. For example, an easily distributed and rapidly degraded soluble substrate, such as lactate, may be combined with a slow-release substrate, such as vegetable oil, as it provides longer term supply of organic carbon. In aquifer systems that are naturally aerobic, an easily distributed and highly degrad‐ able soluble substrate (e.g., ethanol or lactate) can be used to rapidly induce anaerobic, reducing conditions, thus reduce lag phase of anaerobic bacteria. Then a longer lasting, "slowrelease" substrate (e.g., vegetable oil, chitin, or whey) is used to sustain the reaction zone. The combination use of these two types of substrates will minimize the cost of maintaining the

In practice, however, selection of a substrate is often based on contractor experience or familiarity, or as a result of commercial marketing. Substrates are usually selected from a wide variety of available low cost food-grade products such as molasses, vegetable oils, and whey. The potential pitfall of this strategy lies in that ineffective, even inappropriate substrate, may

The underlying principle in determining substrate loading rate is maintaining the balance in a remediation system so that native electron acceptors are fully utilized, while at the same time sufficient electron donor is left in the system to degrade the contaminant mass. To calculate substrate mass required to deplete available electron acceptors flux, substrate composition, stoichiometry, and utilization efficiency of the anticipated degradation reactions need to be known. However, the exact stoichiometric reactions and electron acceptor flux that occur in a site is difficult, if not impractical, to determine [34]. Calculations for substrate demand can be obtained from theoretical hydrogen equivalents resulted from a known mass of substrate versus estimated electron acceptor demand at a site. The electron acceptor demand typically

have been selected in some cases, leading to poor treatment results.

requirements, and cost of implementation (life-cycle cost, including cost of O&M).

form, and in their cost [24, 34].

152 Advances in Bioremediation of Wastewater and Polluted Soil

treatment system [23-24].

**4.2. Organic carbon dosage**

comprises the following three components:


Therefore, it is not easy to have a good estimate on the substrate dosage for a remediation site. On one hand, sufficient carbon is required for contaminant degradation; on the other hand, excessive substrate will not only increase cost, but also may cause secondary problems. In practice, a safety factor of 5 to 20 times of pre-tests may be used to account for the uncertainties at a specific site and to provide for a design contingency. The uncertainties may arise from estimating substrate utilization for alternate electron accepting processes (e.g., methanogene‐ sis or solid-phase alternate electron acceptors), and it may also be caused by the presence of DNAPL or sorbed contaminant mass. Nevertheless, care should be taken to use a loading rate that is not excessive (i.e., use of excessive safety factors) to avoid the creation of low pH conditions or secondary impacts to groundwater quality [24, 34].

Alternatively, the substrate loading rate for soluble substrates can be based on achieving an empirical TOC concentration in groundwater. The volume and strength of substrate are estimated to achieve a particular target level in the treatment area after mixing and dilution. For example, Suthersan et al. [36] suggested that a loading rate between 0.1 and 1 gram of organic carbon per liter of groundwater flux per day is sufficient to create and maintain a reducing reactive zone.

#### **4.3. Additives implementation techniques in bioremediation**

There are a number of system configurations and delivery strategies that can be used to distribute organic substrates in the subsurface. The appropriate technique depends not only on application goal (mass removal or plume containment) but also on the substrates used. The physical nature of substrates dictates the addition technique and potential system configura‐ tions. The frequency of addition is determined by the longevity of the substrate and its ability to supply required substrate and nutrients.

Liquid substrates can be deployed through direct-push or permanent injection wells. Solid substrates are typically placed in trenches or in excavations as backfill in a one-time event using conventional construction techniques. In addition, groundwater recirculation systems, infiltration galleries, and trenches may also be used to deliver substrates to impacted aquifers.

#### *4.3.1. Injection*

Direct injection refers to the process of adding substrates, microorganisms, nutrients, oxidants, or reductants directly into the aquifer at injection points. Installed injection wells or directpush well points are commonly used injection methods to deliver liquid substrates (Figure 1). Injection wells and injection point locations and spacing depend on site geology and hydro‐ geology, aquifer and plume characteristics, and volume of substrates or additives to be injected.

**Figure 1.** Schematic of the use of direct injection system in bioremediation (Adapted from [15])

Direct-push injection is suitable for both soluble and viscous liquid substrates, and is com‐ monly used for shallow groundwater remediation in unconsolidated formations. The appli‐ cation of this technique is constrained by soil characteristics such as grain size or degree of cementation. Gravel and cobbles in sediment inhibit use of direct-push technology. Directpush (e.g., Geoprobe®) injection does not leave well points in place after injection (as opposed to permanent injection wells), and is only practical for slow-release substrates such as HRC®, vegetable oil emulsions, or whey slurries. Direct-push methods have also been used to install semi-permanent well points for short-term injections [23-24].

Permanent injection wells are generally used for soluble substrates where continuous or multiple injections of substrate or recirculation are required. Permanent injection wells are also necessary for sites that cannot use direct-push technology due to depth or soil lithology issues. If screened at appropriate depths and located within appropriate portions of the plume, existing monitoring or extraction wells from previous investigation or remediation activities can also be used for injection [23-24].

Direct injection may be performed through frequent, single-well injections or less-frequent, multiple-well injections with properly spaced injection points. Direct injection is effective at sites with moderate groundwater flow. Sites with very high groundwater flow can be prob‐ lematic due to low cross-gradient distribution of the substrate within the plume. Directinjection approaches are not suitable for highly heterogeneous aquifers because the substrate is not distributed evenly around individual injection points and the contaminant plume.

#### *4.3.2. Recirculation*

physical nature of substrates dictates the addition technique and potential system configura‐ tions. The frequency of addition is determined by the longevity of the substrate and its ability

Liquid substrates can be deployed through direct-push or permanent injection wells. Solid substrates are typically placed in trenches or in excavations as backfill in a one-time event using conventional construction techniques. In addition, groundwater recirculation systems, infiltration galleries, and trenches may also be used to deliver substrates to impacted aquifers.

Direct injection refers to the process of adding substrates, microorganisms, nutrients, oxidants, or reductants directly into the aquifer at injection points. Installed injection wells or directpush well points are commonly used injection methods to deliver liquid substrates (Figure 1). Injection wells and injection point locations and spacing depend on site geology and hydro‐ geology, aquifer and plume characteristics, and volume of substrates or additives to be

**Figure 1.** Schematic of the use of direct injection system in bioremediation (Adapted from [15])

Direct-push injection is suitable for both soluble and viscous liquid substrates, and is com‐ monly used for shallow groundwater remediation in unconsolidated formations. The appli‐ cation of this technique is constrained by soil characteristics such as grain size or degree of

to supply required substrate and nutrients.

154 Advances in Bioremediation of Wastewater and Polluted Soil

*4.3.1. Injection*

injected.

For sites with low groundwater flow rate, recirculation techniques may be required to obtain effective mixing of substrates and contaminated groundwater. Recirculation systems consist of a closed network of extraction wells and injection wells (Figure 2). The recirculation system is designed to hydraulically control substrate transport through the treatment zone. The distance between injection and extraction wells is dictated by groundwater flow velocities, plume size, and bioremediation process kinetics. Excess amendment that is not consumed is extracted at the recovery well and recycled to the injection well. Recirculation approaches may be the only effective method to achieve more uniform distribution of substrates and amend‐ ments at sites that lack significant natural hydraulic gradients [37-38].

Recirculation increases retention time of contaminated groundwater in the treatment zone. Substrate and amendments applied in recirculation systems are more readily controlled and they are distributed through the treatment zone. Recirculation systems can influence a much greater volume of the aquifer, and allow much greater distances between injection and extraction wells; however, continuous operation of a recirculation system requires dedicated equipment and maintenance and thus can be very expensive. In addition, continuous operation creates a potential for biofouling. Alternatively, a recirculation system can be operated in an intermittent fashion, i.e., recirculate for a short time (days to weeks), then shut off for several months, during which time electron donor is consumed and used for contaminant degradation. Intermittent operation of a groundwater recirculation system may be considerably less expensive than continuous recirculation. Periodic operation of a recirculation system will also result in less biofouling of injection wells compared to systems that require continuous recirculation of groundwater and injection of substrates [25].

**Figure 2.** Schematic of a recirculation system in bioremediation (Adapted from [15])

#### *4.3.3. Trenching*

Solid substrate treatment systems are typically deployed in an excavated trench in the form of a permeable reactive barrier (PRB, e.g., biowall) (Figure 3). This treatment method relies on the natural flow of groundwater through solid substrates within the PRB to promote contact with slowly released soluble organic matter. This method is particularly suitable for sites with low permeability. Sometimes, perforated pipes can be laid on the top or bottom of the organic fill material to amend the organic fill material with liquid substrates or other amendments to improve performance [23-24].

Trenches may be installed using either continuous one-pass trenchers designed for installing subsurface utilities or hydraulic excavators. Trench depths are limited by type of equipment used, stability of the formation, and ability of the equipment to excavate the formation. Continuous trenching is not practical in hard and consolidated bedrock [23, 27].
