In this chapter we address several common themes that have emerged from the growing body of bioremediation research and its application in practice. First we review the basic biological principles of bioremediation, including the relationships between contaminant biotransformation and microbial growth. Next, the most com- mon strategies used by microorganisms to attack key structural features of organic contaminants are described, following the approach of Schwarzenbach et al. (2003), so that when unfamiliar contaminant structures are encountered, the reader can anticipate their potential for biotransformation under various conditions. These initial microbial attack mechanisms are then described in the context of the overall biodegradabil- ity of several major classes of contaminants. A key concept is that the biological transformations observed in the laboratory may not be realized in bioremediation endeavors under field conditions. Thus, in the remainder of the chapter we focus primarily on describing (1) factors that may limit biotransformation in the environ- ment, including the genetic capability at a site; (2) various bioremediation strate- gies that have been developed to overcome these limitations; and (3) tools that aid in the design of these technologies, including reaction stoichiometry and microbial kinetics.
8.2 BIODEGRADATION MECHANISMS
Many environmental contaminants are subject to chemical or photochemical reactions. However, biological organisms— particularly microorganisms— play a more important role in the removal of many hazardous organics from the environment. Thermodynam- ically feasible contaminant transformations often do not occur in the absence of a biological catalyst, due to kinetic limitations, but are facilitated by microorganisms via enzymes, which lower the activation energy that must be overcome for a reaction to proceed, and the investment of biochemical energy to convert oxygen and other key coreactants to more reactive forms.
8.2.1 Extent of Biodegradation
Biodegradation is the general term used to describe the biological conversion of organic contaminants to products that are generally lower in free energy (ASCE, 2004). This term is often used loosely and interpreted in various ways. However, it does not imply anything about the extent of contaminant transformation or detoxification. Thus, biodegradation refers to biotransformation reactions that result in only minor changes in contaminant structures, as well as mineralization, which is the conversion of organic compounds into their inorganic constituents (e.g., H2O, CO2, NO3−, SO42−, PO43−,
and Cl−). In some cases, biotransformation reactions generate products that have simi- lar or greater levels of toxicity than those of the parent contaminant. Examples include the conversion of nitroaromatic compounds to more reactive and toxic nitroso and hydroxylamino derivatives (Spain, 1995), and the anaerobic conversion of the sus- pected carcinogen trichloroethene (TCE) to the known carcinogen vinyl chloride (VC) (Freedman and Gossett, 1989). In contrast, the inorganic products of mineralization usu- ally pose no health risks at the concentrations produced by contaminant biodegradation in the environment. Thus, care should be taken in interpreting simple observations of
BIODEGRADATION MECHANISMS 179
parent compound removal in terms of hazard reduction, and biodegradation products should be identified to ensure that bioremediation goals are being met.
8.2.2 Relationship of Biodegradation to Energy Conservation and Growth
The extent of contaminant transformation and detoxification is often related to the ability of an organism to conserve energy and grow via a biodegradation reaction. Complete mineralization of a contaminant is frequently the result of a metabolic process and is linked to energy conservation and biomass synthesis (Alexander, 1981). In contrast, biotransformations that cause only minor changes in contaminant structure are often the result of co-metabolic processes that do not yield free energy or carbon that can be used by the cells carrying out the reactions. Although alternative definitions exist, the term co-metabolism is generally applied to both reactions that occur only in the presence of a growth substrate, as well as reactions that occur without concurrent growth of the organisms carrying out the reactions (Alexander, 1999). Compounds that support microbial growth are known as primary substrates. Co-metabolic substrates are called secondary substrates because they do not support growth. Some compounds can serve as primary substrates at relatively high concentrations but may act as secondary substrates when they are present at levels below Smin, the threshold concentration
needed to supply the organism with sufficient energy for net growth (Rittmann and McCarty, 2001), as discussed below.
Co-metabolic reactions are frequently catalyzed fortuitously by oxygenases or other broad-specificity enzymes. The toluene dioxygenase from Pseudomonas putida F1 is an example of a broad-specificity enzyme and has been shown to act on over 100 substrates with varying structural characteristics (Ellis et al., 2006). The toluene dioxygenase initiates growth of P. putida F1 on toluene, a common groundwater contaminant, by introducing hydroxyl groups into the aromatic ring (Wackett et al., 1988), which leads to the formation of compounds that can be funneled into central metabolic pathways (Seagren and Becker, 2002). The toluene dioxygenase can also catalyze mono- and dioxygenase attack on the mononitrotoluenes in toluene-grown cells of P. putida F1; however, the products of these co-metabolic reactions are not degraded further, and the mononitrotoluenes do not serve as growth substrates for P. putida F1 (Robertson et al., 1992). In this example, toluene serves as a primary substrate for P. putida F1, and the mononitrotoluenes act as secondary substrates.
There are numerous exceptions to these general relationships between the ability of a contaminant to serve as a growth substrate and the extent to which it is trans- formed. For example, co-metabolic reactions sometimes generate products that are not subject to further enzymatic transformations and accumulate in pure cultures but may be acted upon by other species in the environment. The toluene dioxygenase from P. putida F1 co-metabolically converts another common groundwater contami- nant, TCE, to glyoxylate and formate (Li and Wackett, 1992). TCE is not used as a growth substrate by P. putida F1; however, glyoxylate and formate may ultimately be mineralized by other populations within mixed microbial cultures. CO2is also a prod-
uct of aerobic TCE co-metabolism by other species. Thus, co-metabolism sometimes results in partial or complete mineralization of contaminants, particularly in mixed cultures.