There are no documented uses of live genetically modified microorganisms (i.e., microorganisms altered using recombinant DNA) in any commercial project or process for hazardous waste bioremediation. This is certainly true in the United States, and it appears to be the case in the rest of the world as well. There is some anecdotal evidence that specific companies had investigated the use of GMOs in either field remediation or in contained bioreactors (e.g., Envirogen's investigation of recombinant bacteria for TCE degradation in vapor-phase bioreactors; Winter et al. 1989, Glass 1994). In addition, a killed strain of E. coli, engineered to overexpress the enzyme atrazine chlorohydrolase, has been used in the field to remediate atrazine at the site of an accidental spill (Strong et al. 2002; see also "Atrazine Soil Remediation Field Test", at http://biosci.umn.edu/cbri/lisa/web/
index.html). However from the available public record it seems that no living GMOs have ever been used in an actual bioremediation project.
However, there have been two live strains of recombinant microorganisms that have been used in the field for bioremediation research purposes, after having been reviewed and approved by the U.S.
EPA under the TSCA biotechnology regulations. The field trials using these organisms were designed as research experiments, more to validate molecular detection methodology than for any intended remedial purpose.
As shown in Table 3, these are as follows.
64 BIOREMEDIATION OF AQUATIC & TERRESTRIAL ECOSYSTEMS
Gary Sayler of the University of Tennessee and collaborators created a modified strain of Pseudomonas fluorescens HK44 that contained a plasmid encoding genes for naphthalene catabolism as well as an transposon-introduced lux gene under the control of a napththalene catabolic promoter (Ripp et al. 2000, Sayler and Ripp 2000). With both the catabolic genes and the bioluminescent lux gene under the control of the same promoter, this strain could be induced to degrade naphthalene and to bioluminesce by exposure to naphthalene or certain salicylate metabolites. This modified strain was tested in subsurface lysimeters in an experiment at Oak Ridge National Laboratory (ORNL) that lasted from October 1996 to December 1999 (Sayler and Ripp 2000). The microbial inoculant showed enhanced naphthalene gene expression and adequate survival in the lysimeters, however due to heterogeneity in the contaminant concentrations in the lysimeters, it was not possible to make any precise conclusions about the efficacy of using such strains in an actual bioremediation project.
Table 3. Genetically modified microorganisms approved by the U.S. EPA for field testing for bioremediation purposes.
EPA Case Date Institution Microorganism Phenotype Location(s) Number
(TERA unless noted)
PMN 6/28/95 University of Pseudomonas Naphthalene Tennessee P95-1601 Tennessee fluorescens strain degradation
HK44 gene and
bioluminescent reporter gene R98-0004 07/21/98 NEWTEC Pseudomonas Luminesces South
and ORNL putida strain in presence Carolina
RB1500 of TNT
R98-0005 07/21/98 NEWTEC Pseudomonas Fluoeresces South and ORNL putida strain in presence Carolina
RB1501 of TNT
R01-0002 03/28/01 ORNL Pseudomonas Detection California
putida of TNT
R01-0003 04/25/01 ORNL Pseudomonas Detection Ohio
putida of TNT
R01-0004 04/25/01 ORNL Pseudomonas Detection Ohio
putida of TNT
Source: U.S. Environmental Protection Agency, http://www.epa.gov/opptintr/
biotech/submain.htm
The second set of genetically engineered microbial strains used in EPA-approved field testing were created for the purpose of monitoring and detecting contaminants in the field. These are strains of Pseudomonas putida created by Robert Burlage and colleagues of Oak Ridge National Labo-ratory. The parent strains are capable of catabolyzing nitroaromatics like TNT, and Burlage et al. engineered these strains so that a TNT-responsive promoter also controlled expression either of a lux gene or a gene encoding green fluorescent protein. As a result of this engineering, when the microbes are exposed to TNT in the soil, they are expected not only to begin degrading the contaminant, but also to either fluoresce or bioluminesce.
The goal is to use such microorganisms to detect land mines, unexploded ordinance, or other leaking sources of TNT contamination. These strains were first field tested in October 1998 at the National Explosives Waste Technology and Evaluation Center in South Carolina. The recombinant organisms were sprayed onto a site containing simulated mine targets, and then later that day, after dark, the field was surveyed using ultraviolet light to detect areas of microbial activity. According to accounts of the test published on the ORNL website (see "Microbial Minesweepers" at http://www.ornl.gov/info/ornlreview/meas_tech/threat.htm and
"Green Genes: Genetic Technologies for the Environment" at http://www.ornl.gov/info/ornlreview/v32_2_99/green.htm), the bacteria were able to detect the location of all five simulated mine targets in a 300 square meter field. EPA approval was also obtained for subsequent tests at Edwards Air Force Base in California and the Ravenna Army Ammunition Plant in Ohio.
Plans were made for one field test in Europe of a GMO for bioremediation. The research consortium funded by the European Union under the project acronym RHIZODEGRADATION planned to conduct a research field test to document the safety of bioremediation using engineered versions of Pseudomonas fluorescens F113. This strain of P.
fluorescens is a well-known root-colonizing microorganism that has been used in the field. The investigators created a mutant form of F113 with the
‘‘lac’’ZY reporter genes inserted into the chromosome, and then derived a rifampicin-resistant strain by spontaneous mutation. This strain was to be used as a control against another strain, also with a spontaneous rifampicin-resistance mutation, but into which the bph genes from B. cepacia LB400 have been inserted, giving the microbes the abiltiy to use biphenyl as a carbon source. A field test of these two strains was planned to take place at a petroleum hydrocarbon-contaminated site in Arhus, Denmark, however, the test did not receive the needed regulatory approvals and so was never carried out (U. Karlson, personal communication).
66 BIOREMEDIATION OF AQUATIC & TERRESTRIAL ECOSYSTEMS Although there has not yet been a commercial use of a GMO in microbial bioremediation, there is no reason to believe this will not someday occur. The amount of research taking place using recombinant methods to improve biodegradative microorganisms is staggering, and, at least in the U.S., it is clearly possible to conduct outdoor field trials of GMOs with the proper preparation. What has been missing is the commercial and technological need to use a GMO as opposed to an approach involving naturally-occurring microorganisms. Although economic and other factors may yet hold back such proposed uses, others of the commonly perceived barriers may not be significant factors should the right application come along.