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size, strain selection, other strain factors to be taken into account, delivery systems, or the efficacy of freeze-drying (264). Indeed, there is little agreement about the whole contentious issue. As a result, the scientific community in bioremediation is intrigued, whereas the engineering community opinion ranges from suspicion to hostility. An opinion that is often expressed, however, is that there may be no need at all for bioaugmentation in projects such as fuel bioremediation, as there should be indigenous microbes at most sites to deal with fuels, and that the role for bioaugmentation should be the remediation of recalcitrant compounds where there might be so few indigenous strains present that augmentation is necessitated (146). A niche area for bioaugmentation for fuel spills has appeared in the literature: cold climates (e.g., see reference 212), since a great deal of crude oil reserves are extracted in cold regions. Arctic (265) and Antarctic (6, 8) soils are considered to be deficient of microorganisms compared to other soils. Low numbers of hydrocarbon-oxidizing bacteria in pristine cold-climate soils and short summer seasons may limit the spontaneous enrichment of oil-contaminated soils with autochthonous hydrocarbon oxidizers when biostimulation alone is practiced.

As exemplified by tests involving the Exxon Valiez oil spill, a range of strategies and a large number of microorganisms have been used in bioaugmentation, and yet to date there is no guarantee of success (345), especially with commercial products. Mearns (204) found that bioaugmentation with a microbial product did not significantly enhance oil biodégradation in a contaminated marsh in Galveston Bay. Two different microbial products, which exhibited enhanced biodégradation of Alaska North Slope crude oil in shaker flask tests, did not accelerate biodégradation in a field experiment conducted on an oiled beach in Prince William Sound (343). Thouand et al. (315) found that most commercial inocula that they tested lacked any significant ability to degrade crude oil. Venosa et al. (344) also found that an indigenous microbial inoculum did not increase oil biodégradation in a beach environment. Regardless of such equivocal results, many micro-

bial products have been commercialized. Lin et al. (180) concluded that microbial products which are cultured and selected to enhance oil degradation rates have great uncertainties, especially in systems such as wetlands, where hydrocarbon-degrading bacteria are naturally prevalent. They question the value of bioaugmentation for oil spills in light of the fact that addition of microbes had not been demonstrated to be effective in increasing rates of oil degradation and given the high costs of microbial amendments.

The view that increasing the number of microorganisms within the system should increase the rate of removal of contaminants has proven simplistic. Various factors are known to influence the success or failure, but predictability is beyond our means at present. In the case of soil, the milieu is hostile to the introduction of allochthonous microorganisms, and large numbers will never survive unless they have a selective advantage. Critics of bioaugmentation point to the fact that inocula are developed in the laboratory, far from the hostile environments they are intended to compete in (264). The controlling factors are both biotic and abiotic (for a review, see reference 341). The abiotic factors are numerous: they include inappropriate pH or pH shift, UV irradiation, desiccation, and lack of available inorganic nutrients. The biotic factors are more complex and less well understood.

Biotic factors include competition with the indigenous populations and low levels of available carbon substrates (111). A lower threshold substrate concentration for effective bioremed-iation is a controversial area. Sims et al. (295) calculated that at least 150 mg of soil per liter of pore water is required. Another view is that simultaneous utilization of a variety of carbon sources present in low concentrations can be performed until substrate exhaustion at micromolar levels by communities of microorganisms. Indeed, this may be possible within individual microorganisms. For example, the apparent lack of catabolite repression within the rhodococci (354) lends credence to this theory.

FIGURE 5.28 Food web relationships in a percolating filter. After the work ofWheadey (359). Practitioners should remember that adding microorganisms in a bioremediation project is very different from a chemical addition. It has the potential to shift the balance in the food web, so that simply adding more might make more problems, rather than solve any.

FIGURE 5.28 Food web relationships in a percolating filter. After the work ofWheadey (359). Practitioners should remember that adding microorganisms in a bioremediation project is very different from a chemical addition. It has the potential to shift the balance in the food web, so that simply adding more might make more problems, rather than solve any.

In bioaugmentation studies, the predator-prey interaction is often overlooked (Fig. 5.28). For example, in a wastewater bioaugmentation study, Bouchez et al. (44) observed improved performance only during the first day after inoculation, despite inoculation at massive levels. Fluorescent in situ hybridization revealed that the digestive vacuoles of protozoa gave strong signals with a bacterium-specific probe. This provided direct experimental evidence for a warning often expressed over bioaugmentation: high levels of inoculation can lead to an imbalance in the predator-prey interaction, with the wholesale destruction of the inoculum by protozoa. Moreover, the inoculum may also induce strong grazing pressure on other microbial species present, thus creating the risk that the genetic diversity of the site can actually be decreased.

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