Practical Limitations

Given the complexity of the bioremediation process, it is often as critical to know the limitations of the site-specific scenario as it is to know how to apply the technology. Bioremediation is not unlike a golf swing; to play well, good mechanics are everything. Yet, recognizing flaws and adjusting them seems to be an insurmountable task. Environmental factors may stunt or preclude biodégradation even in the presence of engineered organisms. Biodégradation at residual concentrations of the target substrate frequently is a function of the presence of toxicants [34]. During acclimatization and log growth phase, insufficient oxygen and nutrient are the dominant constraints to growth rate. As a target substrate is reduced to levels below 200 mg/kg soil, substrate concentration and substrate inaccessibility may reduce the microbial population to maintenance levels. If the application is being attempted in situ, the transport of oxygen and nutrients to the contaminated zone and the extractability of the affected pore water present yet another set of constraints.

The challenge of effectively introducing nutrients and oxygen to the subsurface can be daunting. Bioslime buildup adjacent to injection wells or drains is a common problem. Subsurface heterogeneities and sorption of nutrients lead to preferential flow pathways for oxygen and nutrient transport, leaving pockets of the contaminated zone virtually untouched. Field experience acquired from responses to tanker and railroad car spills indicates that a practical limit to the size of an in situ treatment system ranges from 3 acres in fine-grained soils (sandy silt) to perhaps 20 acres in porous sands and gravels with native organic contents greater than 3%. Bioremediation in situ for contaminated soil over 5 acres or contaminated groundwater greater than 20 acres in size is not recommended as a sole treatment but may be included in a combination system with such technologies as soil venting, in situ air sparging, or hydraulic containment with above-grade treatment.

Recall that in Section II it was noted that sufficient oxygen was required to commence the catabolism process. Alexander [34] found that operating a system in an oxygen-deficient environment inhibited the metabolism of simple carbohydrates. Aromatic degradation may cease at oxygen concentrations less than 50% of optimum [35], Lee and Ward [36] confirmed these findings by observing a large decay rate for naphthalene, dibenzofuran, and phenanthrene in oxygenated groundwater in comparison to oxygen-depleted water.

Aerobic degradation is not the only pathway for aromatic degradation, however. Mineralization of xylenes in river alluvium under denitrifying conditions has been reported by Kuhn et al. [37]. Benzene, toluene, and alkylbenzenes detected in landfill leachate were mineralized by methanotropic bacteria in a study by Wilson and Rees [38]. Anaerobic decomposition has not been the subject of significant research efforts to date and currently is not being proposed at any of the 80 soil remediation sites under the purview of the EPA [1].

Macronutrient levels should be maintained in excess of optimum whenever practical. In order of importance to growth rate sensitivity, the major macronutrients are nitrogen, phosphorus, potassium, sodium, and calcium.

The effect of declining substrate concentration on biodégradation has been documented in soil and groundwater [39,40]. Kuhlmeier [39] postulated that a threshold may exist for degrading petroleum hydrocarbons in landfarms at 30-70 mg/kg. Functional thresholds for numerous other contaminants such as acetone, bromodichloromethane, and phenols have been raised by others [41-43].

Turning once again to experience acquired from the wastewater industry, another limiting factor can be the amount of inoculum introduced. The theory is that the low cell density that is introduced into soil may not replicate enough to promote effective bioremediation. The effects of predators (protozoa) on the growth of a strain of Pseudomonas bacteria in a sandy loam injected with 50 mg/L methanol was tested. At a cell density of 520 cells/mL, the bacteria declined when protozoa were not inhibited. However, when the protozoa were inhibited by cy-cloheximide and nystatin, the inoculated bacterium multiplied after a 30-h lag period to over 1.7 x 104 cells/mL (Figure 2).

Other factors such as soil permeability, native organic content, sorption, pH, and temperature also play a role in limiting biological degradation rates. Soils exhibiting a permeability greater than 1 x 10~4 cm/s and a total organic content of 3% are more amenable to bioresto-ration. Free product in the subsurface should be removed prior to commencement of biofeasi-bility studies. Sorption has the tendency to concentrate nutrients and can also render the target substrate unavailable to the microbial population. Solubilization techniques such as the addition of surfactants can aid in minimizing this problem.

Soil pH plays a key role in sorption phenomena of ionizable compounds and affects enzyme activity. Several strains of microorganisms known to degrade aromatic hydrocarbons are inhibited at pH values less than 6 [44], and activity approaches zero at soil pH below 5.5. In some cases it is preferable to adjust soil pH where possible to slightly basic. Hambrick et al. [45] observed that mineralization of naphthalene in sediment was faster at pH 8 than at pH 5.

• Bacteria, no inhibitors, 50 m^l ▲ Bacteria, inhibited, 50 ma/I ■ Bacteria, no inhibitors, 100 mg/l □ Bacteria, inhibited, 10(ymgl

Figure 2 Effects of predator organisms on inoculum growth (soil amended with 50 mg/L and 100 mg/L methanol).

100,000

• Bacteria, no inhibitors, 50 m^l ▲ Bacteria, inhibited, 50 ma/I ■ Bacteria, no inhibitors, 100 mg/l □ Bacteria, inhibited, 10(ymgl

A comparison was not made at the less acidic conditions of pH 6 or 6.5, so true optimization could not be determined.

Temperature has a profound effect on microbial activity, particularly in landfarm applications. Several researchers have reported direct relationships between degradation and temperature [39,46,47], Fungi tend to be more temperature-tolerant than bacteria. A general benchmark for temperature thresholds is 55°F for inhibition and 42°F for cell die-off.

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