Bioremediation is generally considered to include a number of specific applications, as summarized below and as described in detail elsewhere in this volume. Most in situ bioremediation methods practiced today rely on the stimulation of indigenous microbial populations at the site of contamination, by addition of appropriate nutrients, principally carbon, oxygen, phosphorus and nitrogen, and by maintaining optimum conditions of pH, moisture and other factors, to trigger increased growth and activity of indigenous biodegradative microorganisms. Applications of this strategy are sometimes referred to by the umbrella term
"biostimulation", with the most commonly practiced variants being:
For in situ treatment of groundwater contamination:
• Bioventing: the injection of oxygen into the unsaturated zone above a water table, in order to stimulate biodegradation by indigenous organisms in the groundwater while also volatilizing ("stripping") certain of the contaminants.
• Biosparging: the injection of oxygen into the saturated zone (i.e., below the water table), so that oxygen bubbles can rise into the unsaturated zone, where natural biodegradation can be stimulated and volatile contaminants stripped.
• Bioslurping: the combination of soil vapor extraction/bioventing with removal of liquid hydrocarbons from the surface of the aquifer (NAPLs -- nonaqueous phase liquids).
For in situ or ex situ treatment of soil contamination:
• Land-farming: the application of soil bioremediation in which adequate oxygenation is ensured by frequent turning or disking of the soil.
• Ex situ or solid-phase bioremediation: in which soil is excavated and placed in a pile where biodegradation is stimulated by addition of nutrients, water, and sometimes added bacterial cultures, surfactants, etc.
In addition to "biostimulation" approaches, soil or groundwater contamination can also be addressed by natural attenuation: the method of allowing contaminant levels to decline over time due to the natural biodegradative capabilities of indigenous microflora. It is important to note that natural attenuation and the various biostimulation approaches share
the common feature that nonindigenous microbial populations are generally not utilized, and that no bacterial cultures are added to the site in any manner.
Remediation technologies in which selected microbial cultures or consortia are introduced to contaminated sites are sometimes referred to as
"bioaugmentation". Bioaugmentation may utilize selected, laboratory-bred microbial strains or microbial consortia that are believed to have enhanced biodegradative capabilities, often against specific compounds or con-taminant categories. Bioaugmentation approaches can be carried out either in situ or ex situ, however bioaugmentation is not widely practiced in commercial remediation. Although there are several reasons for this bias, one major issue is the concern that introduced cultures will not compete well with indigenous species in the environment, and may not survive long enough to carry out their intended purpose.
Another bioremediation (or "biotreatment") application is the use of bioreactors or biofilters in which indigenous or added microorganisms are immobilized on a fixed support, to allow continuous degradation of contaminants. These reactors can be used either with aqueous wastes or slurries or with contaminated vapor phase wastestreams, and in fact microbial biofilters are becoming better accepted within the odor control market and other markets for treatment of contaminated off-gases.
Although most often utilizing indigenous microflora, bioreactors can be used with select, pure microbial cultures, particularly if the reactor is intended for use with a specific contaminant or well-characterized wastestream. One possible use for bioreactors would be the use of microorganisms for biosorption of metals from aqueous wastestreams (discussed below).
Most of the bioremediation technologies described above not only utilize naturally-occurring organisms, but more specifically they rely on species and populations indigenous to the site of contamination. More importantly for the prospects of using GMOs in remediation, these applications generally do not involve the use or introduction of well-defined, selected single-species cultures. It would seem to be an essential prerequisite for the potential use of GMOs in bioremediation that there be accepted, plausible uses for introduction of single-species plants or microbial inocula; otherwise the engineered organisms created in the laboratory would likely not be accepted in the commercial marketplace.
Most microbial inoculants or additives sold for use in bioaugmentation approaches have historically been blends or consortia of microorganisms, purportedly tailored for the types of compounds found in the target waste stream. Initial products were used for municipal waste water treatment or for biotreatment of restaurant grease traps and sewer lines. Several
50 BIOREMEDIATION OF AQUATIC & TERRESTRIAL ECOSYSTEMS companies have sold microbial blends purported to be active against hazardous compounds, including use against industrial effluents and for in situ waste remediation, as well as products rich in lipases, proteases and cellulases for use in activated sludge treatment lagoons or on-line biological reactors for waste water treatment. The most common products for in situ remediation are formulations for degradation of hydrocarbons and petroleum distillates. The earlier of these strains have been used to clean oily bilges in tankers and other ships since the 1960s, and have also attracted attention for their possible usefulness against oil spills on land and sea, although the efficacy of such cultures for this purpose was never proven.
More recently, a number of single-species products have been identified or investigated, and some have been used in commercial remediation.
For example, there are several microbial isolates capable of degrading chlorinated aliphatics. These microbes generally utilize unrelated pathways that fortuitously can metabolize the contaminants of interest.
Trichloroethylene (TCE; the most common pollutant of groundwater) is the most important chlorinated compound that can be biodegraded by such serendipitous pathways. One of the earliest TCE degrading strains to be identified is a pseudomonad (now known as Burkholderia cepacia) named G4 (Shields et al. 1989), that was investigated for commercial use in the early 1990s and continues to be useful in research to this day. Two different strains of Dehalococcoides are now sold commercially for use in bioaugmentation approaches for the dechlorination of TCE or PCE: strain BAV-1, identified at Georgia Tech (He et al. 2003), and now being commercialized by Regenesis Corporation; and KB-1, developed and being sold by DuPont.
Other more recent examples are two microbial cultures that are being used for treatment of methyl tertiary-butyl ether (MTBE). Strain PM1, a member of the >1 subgroup of Proteobacteria, was isolated by Kate Scow and colleagues at UC Davis from a mixed microbial culture originally enriched from a compost biofilter (Hanson et al. 1999). This strain is now being commercialized by Regenesis Corporation for use both for in situ bioaugmentation strategies and also in bioreactors. Salanitro and colleagues isolated a mixed bacterial culture, called BC-1, from chemical plant bioreactor sludge. The culture can be maintained in culture for long periods of time, and can grow on aqueous waste streams with MTBE concentrations of 120-200 ppm. (Salanitro et al. 1994). This strain has been marketed by Shell Global Solutions under the trade name BioRemedy®, and it can be used in the direct inoculation of contaminated groundwater, for intercepting a spreading pollution plume, or for treatment of ground-water in an aboveground reactor.
The fact that most in situ applications of bioremediation involve indigenous microorganisms rather than introduced cultures places a barrier in the path of potential uses of genetically modified microorganisms in bioremediation, that is likely to be a major factor affecting market adoption of GMOs. Many observers feel that a more plausible use for GMOs in remediation will be in bioreactors, designed for use with defined wastestreams. Not only does this avoid the widespread release of the GMO into the environment and avoids the problem of competition with indigenous microflora, but it allows the microorganism to be maintained at controlled temperatures and other growth conditions, and to be used with relatively well-defined wastestreams containing one or a small number of specific contaminants.