Controlled Field Release of a Bioluminescent Genetically Engineered Microorganism for Bioremediation Process Monitoring and Control

Abstract

Pseudomonas fluorescens HK44 represents the first genetically engineered microorganism approved for field testing in the United States for bioremediation purposes. Strain HK44 harbors an introduced lux gene fused within a naphthalene degradative pathway, thereby allowing this recombinant microbe to bioluminesce as it degrades specific polyaromatic hydrocarbons such as naphthalene. The bioremediation process can therefore be monitored by the detection of light. P. fluorescens HK44 was inoculated into the vadose zone of intermediate-scale, semicontained soil lysimeters contaminated with naphthalene, anthracene, and phenanthrene, and the population dynamics were followed over an approximate 2-year period in order to assess the long-term efficacy of using strain HK44 for monitoring and controlling bioremediation processes. Results showed that P. fluorescens HK44 was capable of surviving initial inoculation into both hydrocarbon contaminated and uncontaminated soils and was recoverable from these soils 660 days post inoculation. It was also demonstrated that strain HK44 was capable of generating bioluminescence in response to soil hydrocarbon bioavailability. Bioluminescence approaching 166 000 counts/s was detected in fiber optic-based biosensor devices responding to volatile polyaromatic hydrocarbons, while a portable photomultiplier module detected bioluminescence at an average of 4300 counts/s directly from soil-borne HK44 cells within localized treatment areas. The utilization of lux-based bioreporter microorganisms therefore promises to be a viable option for in situ determination of environmental contaminant bioavailability and biodegradation process monitoring and control.

Synthetic Genomics and Synthetic Biology Applications Between Hopes and Concerns

“…The introduced genetic circuits can vary greatly in complexity, however, and examples comprising natural sensor proteins [5,50] can be distinguished from those that involve "non-natural" sensor proteins with novel specificities, generated by directed evolution [51] or computational design [52]. Though involving few components, the approaches involving "non-natural" sensor proteins as well as the recent integration of a synthetic riboswitch capable of controlling migration of green fluorescent protein expressing bacteria towards the pesticide atrazine (followed by its degradation) [53] (see Fig.…”

Grand Challenges for Biological and Environmental Research: A Long-Term Vision

“…These "realistic" mesocosms would provide a suitable environment for testing synthetic microbial systems in a controlled setting. BER has supported pioneering the use of designed organisms to enhance process understanding at the field scale (Ripp et al 2000). Recent advances in genetic containment and control strategy greatly December 2010…”

Ameliorating Risk: Culturable and Metagenomic Monitoring of the 14 Year Decline of a Genetically Engineered Microorganism at a Bioremediation Field Site

“…The first United States Environmental Protection Agency (EPA) approved application of a genetically engineered microorganism for bioremediation purposes occurred in 1996 at an intermediate scale (4 m deep × 2.5 m diameter), semi-contained, and replicated soil lysimeter site at the Oak Ridge National Laboratory, Oak Ridge, Tennessee [1]. This organism, Pseudomonas fluorescens HK44, was capable of degrading polycyclic aromatic hydrocarbons (PAHs) and contained the genes for bioluminescence (luxCDABE) downstream of the salicylate inducible nahR promoter on a plasmid (pUTK21) construct [2].…”

“…Strain HK44 was sprayed on to layered 10 cm soil lifts 1 m deep artificially contaminated with a PAH mixture of naphthalene, anthracene, and phenanthrene [4]. Bioluminescence from these soil-borne bioreporters was then monitored using fiber optic and photomultiplier tube (PMT) interfaces over the ensuing two years to establish the potential for bioreporters to serve as bioremediation process monitoring and control tools [1]. Ancillary to this was the critical question of whether genetically engineered microorganisms like HK44 could even survive under conventional environmental pressures for this length of time due to the extra metabolic burdens and lack of fitness that these recombinant microorganisms perceivably bear.…”

“…Thus, HK44 populations were monitored during the initial two years of the release using selective plating and colony hybridization techniques, and it was shown that population concentrations decreased from an initial inoculum level of approximately 1 × 106 cfu/g soil to a final maintenance concentration of approximately 1 × 103 cfu/g soil. At these final low populations, however, HK44 populations remained in a physiological state conducive to reactivation and regrowth, and were still capable of generating bioluminescence when exposed to supplemental PAH contaminants and an inorganic nutrient amendment [1]. Based on this interesting outcome, it was decided that the lysimeter facility remain untouched and accessible for future longer-term population monitoring activities, and were thus covered with steel lids and remain today as a vegetation-free, desiccated soil ecosystem.…”