Responses of three grass species to creosote during phytoremediation.

Phytoremediation of creosote-contaminated soil was monitored in the presence of Tall fescue, Kentucky blue grass, or Wild rye. For all three grass species, plant growth promoting rhizobacteria (PGPR) were evaluated for plant growth promotion and protection of plants from contaminant toxicity. A number of parameters were monitored including plant tissue water content, root growth, plant chlorophyll content and the chlorophyll a/b ratio. The observed physiological data indicate that some plants mitigated the toxic effects of contaminants. In addition, in agreement with our previous experiments reported in the accompanying paper (Huang, X.-D., El-Alawi, Y., Penrose, D.M., Glick, B.R., Greenberg, B.M., 2004. A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soil. Environ. Poll. doi: 10.1016/j.envpol.2003.09.031), PGPR were able to greatly enhance phytoremediation. PGPR accelerated plant growth, especially roots, in heavily contaminated soils, diminishing the toxic effects of contaminants to plants. Thus, the increased root biomass in PGPR-treated plants led to more effective remediation.

A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soils.

To improve phytoremediation processes, multiple techniques that comprise different aspects of contaminant removal from soils have been combined. Using creosote as a test contaminant, a multi-process phytoremediation system composed of physical (volatilization), photochemical (photooxidation) and microbial remediation, and phytoremediation (plant-assisted remediation) processes was developed. The techniques applied to realize these processes were land-farming (aeration and light exposure), introduction of contaminant degrading bacteria, plant growth promoting rhizobacteria (PGPR), and plant growth of contaminant-tolerant tall fescue (Festuca arundinacea). Over a 4-month period, the average efficiency of removal of 16 priority PAHs by the multi-process remediation system was twice that of land-farming, 50% more than bioremediation alone, and 45% more than phytoremediation by itself. Importantly, the multi-process system was capable of removing most of the highly hydrophobic, soil-bound PAHs from soil. The key elements for successful phytoremediation were the use of plant species that have the ability to proliferate in the presence of high levels of contaminants and strains of PGPR that increase plant tolerance to contaminants and accelerate plant growth in heavily contaminated soils. The synergistic use of these approaches resulted in rapid and massive biomass accumulation of plant tissue in contaminated soil, putatively providing more active metabolic processes, leading to more rapid and more complete removal of PAHs.

Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria.

One of the major mechanisms utilized by plant growth-promoting rhizobacteria (PGPR) to facilitate plant growth and development is the lowering of ethylene levels by deamination of 1-aminocyclopropane-1-carboxylic acid (ACC) the immediate precursor of ethylene in plants. The enzyme catalysing this reaction, ACC deaminase, hydrolyses ACC to alpha-ketobutyrate and ammonia. Several bacterial strains that can utilize ACC as a sole source of nitrogen have been isolated from rhizosphere soil samples. All of these strains are considered to be PGPR based on the ability to promote canola seedling root elongation under gnotobiotic conditions. The treatment of plant seeds or roots with these bacteria reduces the amount of ACC in plants, thereby lowering the concentration of ethylene. Here, a rapid procedure for the isolation of ACC deaminase-containing bacteria, a root elongation assay for evaluating the effects of selected bacteria on root growth, and a method of assessing bacterial ACC deaminase activity are described in detail. This should allow researchers to readily isolate new PGPR strains adapted to specific environments.

Strategies used by rhizobia to lower plant ethylene levels and increase nodulation.

Agriculture depends heavily on biologically fixed nitrogen from the symbiotic association between rhizobia and plants. Molecular nitrogen is fixed by differentiated forms of rhizobia in nodules located on plant roots. The phytohormone, ethylene, acts as a negative factor in the nodulation process. Recent discoveries suggest several strategies used by rhizobia to reduce the amount of ethylene synthesized by their legume symbionts, decreasing the negative effect of ethylene on nodulation. At least one strain of rhizobia produces rhizobitoxine, an inhibitor of ethylene synthesis. Active 1-aminocyclopropane-1-carboxylate (ACC) deaminase has been detected in a number of other rhizobial strains. This enzyme catalyzes the cleavage of ACC to alpha-ketobutyrate and ammonia. It has been shown that the inhibitory effect of ethylene on plant root elongation can be reduced by the activity of ACC deaminase.