Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Oct;63(10):4053–4060. doi: 10.1128/aem.63.10.4053-4060.1997

Development and characterization of a whole-cell bioluminescent sensor for bioavailable middle-chain alkanes in contaminated groundwater samples.

P Sticher 1, M C Jaspers 1, K Stemmler 1, H Harms 1, A J Zehnder 1, J R van der Meer 1
PMCID: PMC168716  PMID: 9327569

Abstract

A microbial whole-cell biosensor was developed, and its potential to measure water-dissolved concentrations of middle-chain-length alkanes and some related compounds by bioluminescence was characterized. The biosensor strain Escherichia coli DH5 alpha(pGEc74, pJAMA7) carried the regulatory gene alkS from Pseudomonas oleovorans and a transcriptional fusion of PalkB from the same strain with the promoterless luciferase luxAB genes from Vibrio harveyi on two separately introduced plasmids. In standardized assays, the biosensor cells were readily inducible with octane, a typical inducer of the alk system. Light emission after induction periods of more than 15 min correlated well with octane concentration. In well-defined aqueous samples, there was a linear relationship between light output and octane concentrations between 24 and 100 nM. The biosensor responded to middle-chain-length alkanes but not to alicyclic or aromatic compounds. In order to test its applicability for analyzing environmentally relevant samples, the biosensor was used to detect the bioavailable concentration of alkanes in heating oil-contaminated groundwater samples. By the extrapolation of calibrated light output data to low octane concentrations with a hyperbolic function, a total inducer concentration of about 3 nM in octane equivalents was estimated. The whole-cell biosensor tended to underestimate the alkane concentration in the groundwater samples by about 25%, possibly because of the presence of unknown inhibitors. This was corrected for by spiking the samples with a known amount of an octane standard. Biosensor measurements of alkane concentrations were further verified by comparing them with the results of chemical analyses.

Full Text

The Full Text of this article is available as a PDF (291.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blouin K., Walker S. G., Smit J., Turner R. Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes. Appl Environ Microbiol. 1996 Jun;62(6):2013–2021. doi: 10.1128/aem.62.6.2013-2021.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  3. Burlage R. S., Sayler G. S., Larimer F. Monitoring of naphthalene catabolism by bioluminescence with nah-lux transcriptional fusions. J Bacteriol. 1990 Sep;172(9):4749–4757. doi: 10.1128/jb.172.9.4749-4757.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohn D. H., Mileham A. J., Simon M. I., Nealson K. H., Rausch S. K., Bonam D., Baldwin T. O. Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase. J Biol Chem. 1985 May 25;260(10):6139–6146. [PubMed] [Google Scholar]
  5. DiMarco A. A., Averhoff B., Ornston L. N. Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus. J Bacteriol. 1993 Jul;175(14):4499–4506. doi: 10.1128/jb.175.14.4499-4506.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Efroymson R. A., Alexander M. Biodegradation by an arthrobacter species of hydrocarbons partitioned into an organic solvent. Appl Environ Microbiol. 1991 May;57(5):1441–1447. doi: 10.1128/aem.57.5.1441-1447.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eggink G., Engel H., Meijer W. G., Otten J., Kingma J., Witholt B. Alkane utilization in Pseudomonas oleovorans. Structure and function of the regulatory locus alkR. J Biol Chem. 1988 Sep 15;263(26):13400–13405. [PubMed] [Google Scholar]
  8. Eggink G., Lageveen R. G., Altenburg B., Witholt B. Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli. J Biol Chem. 1987 Dec 25;262(36):17712–17718. [PubMed] [Google Scholar]
  9. Engebrecht J., Simon M., Silverman M. Measuring gene expression with light. Science. 1985 Mar 15;227(4692):1345–1347. doi: 10.1126/science.2983423. [DOI] [PubMed] [Google Scholar]
  10. Harms H. Bacterial Growth on Distant Naphthalene Diffusing through Water, Air, and Water-Saturated and Nonsaturated Porous Media. Appl Environ Microbiol. 1996 Jul;62(7):2286–2293. doi: 10.1128/aem.62.7.2286-2293.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heitzer A., Malachowsky K., Thonnard J. E., Bienkowski P. R., White D. C., Sayler G. S. Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium. Appl Environ Microbiol. 1994 May;60(5):1487–1494. doi: 10.1128/aem.60.5.1487-1494.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heitzer A., Webb O. F., Thonnard J. E., Sayler G. S. Specific and quantitative assessment of naphthalene and salicylate bioavailability by using a bioluminescent catabolic reporter bacterium. Appl Environ Microbiol. 1992 Jun;58(6):1839–1846. doi: 10.1128/aem.58.6.1839-1846.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnston T. C., Thompson R. B., Baldwin T. O. Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase. J Biol Chem. 1986 Apr 15;261(11):4805–4811. [PubMed] [Google Scholar]
  15. King J. M., Digrazia P. M., Applegate B., Burlage R., Sanseverino J., Dunbar P., Larimer F., Sayler G. S. Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science. 1990 Aug 17;249(4970):778–781. doi: 10.1126/science.249.4970.778. [DOI] [PubMed] [Google Scholar]
  16. Korpela M., Mäntsälä P., Lilius E. M., Karp M. Stable-light-emitting Escherichia coli as a biosensor. J Biolumin Chemilumin. 1989 Jul;4(1):551–554. doi: 10.1002/bio.1170040172. [DOI] [PubMed] [Google Scholar]
  17. Miller R. M., Bartha R. Evidence from liposome encapsulation for transport-limited microbial metabolism of solid alkanes. Appl Environ Microbiol. 1989 Feb;55(2):269–274. doi: 10.1128/aem.55.2.269-274.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Selifonova O. V., Eaton R. W. Use of an ipb-lux Fusion To Study Regulation of the Isopropylbenzene Catabolism Operon of Pseudomonas putida RE204 and To Detect Hydrophobic Pollutants in the Environment. Appl Environ Microbiol. 1996 Mar;62(3):778–783. doi: 10.1128/aem.62.3.778-783.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Selifonova O., Burlage R., Barkay T. Bioluminescent sensors for detection of bioavailable Hg(II) in the environment. Appl Environ Microbiol. 1993 Sep;59(9):3083–3090. doi: 10.1128/aem.59.9.3083-3090.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shingler V., Moore T. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. J Bacteriol. 1994 Mar;176(6):1555–1560. doi: 10.1128/jb.176.6.1555-1560.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stewart G. S. In vivo bioluminescence: new potentials for microbiology. Lett Appl Microbiol. 1990 Jan;10(1):1–8. doi: 10.1111/j.1472-765x.1990.tb00082.x. [DOI] [PubMed] [Google Scholar]
  22. Stucki G., Alexander M. Role of dissolution rate and solubility in biodegradation of aromatic compounds. Appl Environ Microbiol. 1987 Feb;53(2):292–297. doi: 10.1128/aem.53.2.292-297.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thomas J. M., Yordy J. R., Amador J. A., Alexander M. Rates of dissolution and biodegradation of water-insoluble organic compounds. Appl Environ Microbiol. 1986 Aug;52(2):290–296. doi: 10.1128/aem.52.2.290-296.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Van Dyk T. K., Majarian W. R., Konstantinov K. B., Young R. M., Dhurjati P. S., LaRossa R. A. Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. Appl Environ Microbiol. 1994 May;60(5):1414–1420. doi: 10.1128/aem.60.5.1414-1420.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Watkinson R. J., Morgan P. Physiology of aliphatic hydrocarbon-degrading microorganisms. Biodegradation. 1990;1(2-3):79–92. doi: 10.1007/BF00058828. [DOI] [PubMed] [Google Scholar]
  26. Wood K. V., Gruber M. G. Transduction in microbial biosensors using multiplexed bioluminescence. Biosens Bioelectron. 1996;11(3):207–214. doi: 10.1016/0956-5663(96)88407-7. [DOI] [PubMed] [Google Scholar]
  27. de Lorenzo V., Fernández S., Herrero M., Jakubzik U., Timmis K. N. Engineering of alkyl- and haloaromatic-responsive gene expression with mini-transposons containing regulated promoters of biodegradative pathways of Pseudomonas. Gene. 1993 Aug 16;130(1):41–46. doi: 10.1016/0378-1119(93)90344-3. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES