Illuminating environmental monitoring with living bioreporters Steven Ripp The University of Tennessee Center for Environmental Biotechnology

~27,000 students from over 100 different countries

Analyte Promoter Reporter Gene Transcription mRNA Translation Signal The anatomy of a bioreporter 

Reporter systems Reporter geneDisadvantage Chloramphenicol acetyltransferase (CAT) Often employs radioisotopes, requires the addition of substrate, requires separation of substrate and product  -galactosidase Endogenous activity, requires the addition of substrate AequorinRequires the addition of substrate and high Ca 2+ Firefly luciferase (luc)Requires the addition of substrate Green Fluorescent Protein (GFP) Requires activation by external source Bacterial luciferase (lux)Requires oxygen, discrete temperature range

The attributes of bacterial luciferase (lux) Autonomous response No user interaction required Repeatable, re-usable, nondestructive Near real-time response Easily measured output (light) with no requirement for excitation source But it is a living system ATP, O 2, NADPH Temperature, pH, salinity extremes Target toxicity But living is good bioavailability BioreporterSample

lux-based bioreporter assays ‘Lights-off’ assays Bioreporter continuously produces bioluminescence (constitutive lux reporter gene system) A decrease in bioluminescence upon exposure to a chemical indicates toxicity i.e., the Microtox ® assay ‘Lights-on’ assays Bioreporter generates bioluminescence only when induced by a specific compound or family of compounds (inducible lux reporter gene system)

Bioluminescent bioreporter chemical targets Metals Cadmium (4 h, 19 mg/kg) Chromate (1 h, 10 µM) Cobalt (not specified, 2 mM) Copper (1 h, 1 µM) Heavy metals Iron (Hours, 10 nM) Lead (4 h, 4 g/kg) Mercury (70 min, nM) Nickel (not specified, 0.3 mM) Zinc (4 h, 0.5 µM) Food/water/air quality Aflatoxin (45 min, 1.2 ppm) Ammonia (30 min, 20 µM) Estrogens/Androgens (1-4 h, M) Histamine (30 min, 20 ppm) Nitrate (4 h, 0.05 µM) Tetracycline (40 min, 5 ng/L) Organics 2,4-Dichlorophenol (2 h, 50 mg/L) 3-Xylene (Hours, 3 µM) 4-Chlorobenzoate (1 h, 380 µM) 4-Nitrophenol (2 h, 0.25 mg/L) BTEX (1-4 h, 0.03 mg/L) Chloroform (2 h, 300 mg/L) Dichloromethane (3 h, saturated) Hydrogen peroxide (20 min, 0.1 mg/L) Isopropyl benzene (1-4 h, 1 µM) Naphthalene (8-24 min, 12 µM) Organic peroxides (20 min, not specified) PCBs (1-3 h, 0.8 µM) p-chlorobenzoic acid (40 min, 0.06 g/L) p-cymene (<30 min, 60 ppb) Phenol (2 h, 16 mg/L) Salicylate (15 min, 36 µM) Trichloroethylene (1-1.5 h, 5 µM)

The bioreporter Pseudomonas fluorescens HK44 Upper PathwayLower Pathway Naphthalene  SalicylateSalicylate  2-oxo-4-hydroxypentanoate ABFCEDR GHINLJK ABFCEDR G lux cassette Naphthalene  SalicylateSalicylate  2-oxo-4-hydroxypentanoate X Wild-type Bioreporter

Bioreporter HK44 as a bioremediation process monitoring and control tool

Bioremediation monitoring with P. fluorescens HK44 Time 0 2 years later Naphthalene (ppm) Bioluminescence Fiber Optic Cable Encapsulated HK44 Cells Porous Metal Housing in situ soil bioluminescence

Long-term (14 year) bioreporter survival LysimeterConditions Number of samples Quantitative PCR copies/g (% of positive samples) nahAtetAluxA 1 PAH contaminated + HK44 36 Not detected 4455 (17%) Not detected 2 PAH contaminated + HK44 36 Not detected 872 (17%) 2052 (17%) Chemically contaminated lysimeters (total heterotrophs) Control lysimeters (total heterotrophs) Chemically contaminated lysimeters (tetracycline resistant) Control lysimeters (tetracycline resistant)

Monitoring groundwater contamination at a U.S. Air Force Base Pseudomonas fluorescens TVA8 bioreporter specific for BTEX (benzene, toluene, ethylbenzene, xylene) jet fuel components

Upgradient Downgradient Groundwater Flow Distance (meters) Depth (meters above sea level) m 0 3 m

BTEX profiles (ppm) Meters Bioluminescence GC/MS Source trench

Evanescent optical fiber sensors Optical claddings consisting of uniquely ‘colored’ optical bioreporters Fiber core Fiber length (m) Light attenuation (spectrum) Backscatter light Cladding

Other environmental bioreporter sensing applications On-line detection of wastewater treatment upsets Remote detection of microbial ‘sick building syndrome’ contaminants On-board UAV penetration through aerosol clouds Water toxicity monitoring

Using reporter bacteriophage (bacterial viruses) to target bacterial pathogens Rinsate

CCD imaging of bioluminescence output E. coli cfu/mL A bioluminescent phage assay for Escherichia coli

hour preincubation A bioluminescent phage assay for Escherichia coli

Optical Application Specific Integrated Circuits (BBICs) Encapsulated bioluminescent bioreporters Photodetectors Signal processing circuitry Opaque porous barrier

Whole cell LuxArray Analyte flow Removable, reusable circuit board (underneath) 96 distinct cellular reporter fluid reservoirs Flow- through output 9 cm 4 cm Delrin ® housing Reservoir layer Nanoplotted cellular reporter membrane Printed circuit board

Obstacles to overcome We are not bacteria! The effect of a chemical on a bacterial cell does not adequately profile human/animal toxicity ‘Real-world’ applications are rare Regulatory agencies reluctant to adopt Public perception of risk difficult to overcome Short selling the technology Bioreporters complement but do not replace conventional analytical chemical detection methods Engineered microorganisms cannot survive in the environment (or can they?)

Acknowledgements The National Science Foundation, grants # CBET and DBI BioTech Inc. The Center for Environmental Biotechnology Dr. Gary Sayler Dr. Tingting Xu Dr. Dan Close Pat Jegier Dean Webb Alexandra Rogers Clara Beasley