Analytical and Bioanalytical Chemistry

, Volume 400, Issue 4, pp 1093–1104 | Cite as

In situ detection of aromatic compounds with biosensor Pseudomonas putida cells preserved and delivered to soil in water-soluble gelatin capsules

  • Aitor de las Heras
  • Víctor de LorenzoEmail author
Original Paper


While many types of bacteria have been engineered to produce an optical output in response to given analytes in a culture, their use for extensive, in situ monitoring of distinct chemical species in soil is hampered by a dearth of practicable spreading schemes. In this work, we report and validate a comprehensive system for the long-term preservation of Pseudomonas putida cells genetically designed for biosensing benzene, toluene, ethylbenzene, and xylenes (BTEX) in soil, along with a procedure to formulate, spread, and vigorously activate such bacteria at the desired site and occasion. To this end, various known lyoprotectants were tested for promoting the long-term maintenance of biosensor cells with quite variable outcomes. While a formulation of inositol and maltodextrines was optimal for preservation of freeze-dried BTEX-sensing bacteria, adsorption of P. putida cells to corncob powder (an abundant residue of the corn industry) endowed the resulting material with a lasting viability at ambient conditions. In any case, the thereby preserved bacterial biomass acquired physical and mechanical properties adequate for formulating the biosensor agent in water-soluble but otherwise hard dry gelatine capsules with a long shelf life. When such capsules were spread in a soil microcosm and subsequently liquefied with water or high humidity, the released microorganisms formed spots that gave an intense luminiscent signal upon exposure to effectors of the sensor circuit implanted in the chromosome of the P. putida strain. We argue that the procedures described here can facilitate implementation of wide-area biological detection strategies for revealing the location of toxic or perilous chemicals.


Pseudomonas putida XylR Biosensors Desiccation Encapsulation 



We thank JR van der Meer (University of Lausanne, Switzerland) for his gift of corncob and J. Pliego (OMFE) for providing hard gelatin capsules. We also thank Rob Cain for his comments and Esther Fernández for technical help. This work was defrayed by generous grants of the CONSOLIDER program of the Spanish Ministry of Science and Innovation, by the TARPOL, BACSINE, and MICROME Contracts of the EU and by funds of the Autonomous Community of Madrid.


  1. 1.
    Cases I, de Lorenzo V (2005) Promoters in the environment: transcriptional regulation in its natural context. Nat Rev Microbiol 3:105–118CrossRefGoogle Scholar
  2. 2.
    Tropel D, van der Meer JR (2004) Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500CrossRefGoogle Scholar
  3. 3.
    Girotti S, Ferri EN, Fumo MG, Maiolini E (2008) Monitoring of environmental pollutants by bioluminescent bacteria. Anal Chim Acta 608:2–29CrossRefGoogle Scholar
  4. 4.
    Dawson JJ, Iroegbu CO, Maciel H, Paton GI (2008) Application of luminescent biosensors for monitoring the degradation and toxicity of btex compounds in soils. J Appl Microbiol 104:141–151Google Scholar
  5. 5.
    Ron EZ (2007) Biosensing environmental pollution. Curr Opin Biotechnol 18:252–256CrossRefGoogle Scholar
  6. 6.
    van der Meer JR, Tropel D, Jaspers M (2004) Illuminating the detection chain of bacterial bioreporters. Environ Microbiol 6:1005–1020CrossRefGoogle Scholar
  7. 7.
    Bjerketorp J, Håkansson S, Belkin S, Jansson JK (2006) Advances in preservation methods: keeping biosensor microorganisms alive and active. Curr Opin Biotechnol 17:1–7CrossRefGoogle Scholar
  8. 8.
    Marqués S, Aranda-Olmedo I, Ramos JL (2006) Controlling bacterial physiology for optimal expression of gene reporter constructs. Curr Opin Biotechnol 17:50–56CrossRefGoogle Scholar
  9. 9.
    Batzias F, Siontorou CG (2007) A novel system for environmental monitoring through a cooperative/synergistic scheme between bioindicators and biosensors. J Environ Manage 82:221–239CrossRefGoogle Scholar
  10. 10.
    de Las HA, Carreño CA, de Lorenzo V (2008) Stable implantation of orthogonal sensor circuits in gram-negative bacteria for environmental release. Env Microbiol 10:3305–3316CrossRefGoogle Scholar
  11. 11.
    Smith R, D’souza N, Nicklin S (2008) A review of biosensors and biologically-inspired systems for explosives detection. Analyst 133:571CrossRefGoogle Scholar
  12. 12.
    Galvão TC, de Lorenzo V (2006) Transcriptional regulators à la carte: engineering new effector specificities in bacterial regulatory proteins. Curr Opin Biotechnol 17:34–42CrossRefGoogle Scholar
  13. 13.
    Gu MB, Choi SH, Kim SW (2001) Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring. J Biotechnol 88:95–105CrossRefGoogle Scholar
  14. 14.
    Meurer H, Wehner H, Schillberg S, Hund-Rinke K, Kühn C, Raven N, Wirtz T (2010) An emerging remote sensing technology and its potential impact on mine action. In: Jungwirth O (ed) Proceedings of the international symposium on humanitarian demining. HCR-CTRO Zagreb (Croatia). pp. 66–70Google Scholar
  15. 15.
    Applegate BM, Kehrmeyer SR, Sayler GS (1998) A chromosomally based tod-luxCDABE whole-cell reporter for benzene, toluene, ethybenzene, and xylene (btex) sensing. Appl Environ Microbiol 64:2730–2735Google Scholar
  16. 16.
    Hay AG, Rice JF, Applegate BM, Bright NG, Sayler GS (2000) A bioluminescent whole-cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2, 4-dichlorophenol in soil. Appl Environ Microbiol 66:4589–4594CrossRefGoogle Scholar
  17. 17.
    Willardson BM, Wilkins JF, Rand TA, Schupp JM, Hill KK, Keim P, Jackson PJ (1998) Development and testing of a bacterial biosensor for toluene-based environmental contaminants. Appl Environ Microbiol 64:1006–1012Google Scholar
  18. 18.
    van der Meer JR, Belkin S (2010) Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8:511–522CrossRefGoogle Scholar
  19. 19.
    Date A, Pasini P, Daunert S (2007) Construction of spores for portable bacterial whole-cell biosensing systems. Anal Chem 79:9391–9397CrossRefGoogle Scholar
  20. 20.
    Hakkila K, Green T, Leskinen P, Ivask A, Marks R, Virta M (2004) Detection of bioavailable heavy metals in eilatox-oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips. J Appl Toxicol 24:333–342CrossRefGoogle Scholar
  21. 21.
    Morgan CA, Herman N, White PA, Vesey G (2006) Preservation of micro-organisms by drying; a review. J Microbiol Meth 66:183–193CrossRefGoogle Scholar
  22. 22.
    Miyamoto-Shinohara Y, Sukenobe J, Imaizumi T (2006) Survival curves for microbial species stored by freeze-drying. Cryobiology 52:27–32CrossRefGoogle Scholar
  23. 23.
    Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P (2003) Effect of various growth media upon survival during storage of freeze-dried Enterococcus faecalis and Enterococcus durans. J Appl Microbiol 94:947–952CrossRefGoogle Scholar
  24. 24.
    Muñoz-Rojas J, Bernal P, Duque E, Godoy P (2006) Involvement of cyclopropane fatty acids in the response of Pseudomonas putida KT2440 to freeze-drying. Appl Environ Microbiol 72:472–477CrossRefGoogle Scholar
  25. 25.
    Zhao G, Zhang G (2005) Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying. J Appl Microbiol 99:333–338CrossRefGoogle Scholar
  26. 26.
    Mertens B, Boon N, Verstraete W (2006) Slow-release inoculation allows sustained biodegradation of gamma-hexachlorocyclohexane. Appl Environ Microbiol 72:622–627CrossRefGoogle Scholar
  27. 27.
    Elsas JD, Trevors JT, Jain D, Wolters AC (1992) Survival of, and root colonization by, alginate-encapsulated Pseudomonas fluorescens cells following introduction into soil. Biol Fertil Soils 14:14–22CrossRefGoogle Scholar
  28. 28.
    Hall McLoughlin AJ, Leung KT, Trevors JT, Lee HY (1998) Transport and survival of alginate-encapsulated and free lux-lac marked Pseudomonas aeruginosa ug2lr cells in soil. FEMS Microbiol Ecol 26:51–61CrossRefGoogle Scholar
  29. 29.
    Garmendia J, Devos D, Valencia A, de Lorenzo V (2001) A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors. Mol Microbiol 42:47–59CrossRefGoogle Scholar
  30. 30.
    Galvão TC, Mencía M, de Lorenzo V (2007) Emergence of novel functions in transcriptional regulators by regression to stem protein types. Mol Microbiol 65:907–919CrossRefGoogle Scholar
  31. 31.
    Regenhardt D, Heuer H, Heim S, Fernandez DU, Strömpl C, Moore ER, Timmis KN (2002) Pedigree and taxonomic credentials of pseudomonas putida strain KT2440. Environ Microbiol 4:912–915CrossRefGoogle Scholar
  32. 32.
    Bagdasarian M, Timmis KN (1982) Host: vector systems for gene cloning in pseudomonas. Curr Top Microbiol Immunol 96:47–67Google Scholar
  33. 33.
    Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VA, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Chris Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen JA, Timmis KN, Düsterhöft A, Tümmler B, Fraser CM (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808CrossRefGoogle Scholar
  34. 34.
    Garmendia J, de las Heras A, Galvao TC, Lorenzo VD (2008) Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes. Micro Biotech 1:236–246CrossRefGoogle Scholar
  35. 35.
    Fernandez S, de Lorenzo V, Perez-Martin J (1995) Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains. Mol Microbiol 16:205–213CrossRefGoogle Scholar
  36. 36.
    Bjarnason J, Southward CM, Surette MG (2003) Genomic profiling of iron-responsive genes in Salmonella enterica serovar typhimurium by high-throughput screening of a random promoter library. J Bacteriol 185:4973–4982CrossRefGoogle Scholar
  37. 37.
    de Lorenzo V, Timmis KN (1994) Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235:386–405CrossRefGoogle Scholar
  38. 38.
    Lambertsen L, Sternberg C, Molin S (2004) Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ Microbiol 6:726–732CrossRefGoogle Scholar
  39. 39.
    McKown RL, Orle KA, Chen T, Craig NL (1988) Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion. J Bacteriol 170:352–358Google Scholar
  40. 40.
    Peters JE, Craig NL (2001) Tn7: smarter than we thought. Nat Rev Mol Cell Biol 2:806–814CrossRefGoogle Scholar
  41. 41.
    de Lorenzo V, Cases I, Herrero M, Timmis KN (1993) Early and late responses of tol promoters to pathway inducers: identification of postexponential promoters in pseudomonas putida with lacZ-tet bicistronic reporters. J Bacteriol 175:6902–6907Google Scholar
  42. 42.
    Olivella L, Figueras M, Fraile J, Vilanova M, Ginebreda A, Barcelo D (2002) Fate of MTBE and DCPD compounds relative to BTEX in gasoline-contaminated aquifers. Scientific World Journal 2:1108–1114Google Scholar
  43. 43.
    Ramos JL, Marques S (1997) Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol 51:341–373CrossRefGoogle Scholar
  44. 44.
    Abril MA, Michan C, Timmis KN, Ramos JL (1989) Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol 171:6782–6790Google Scholar
  45. 45.
    Kim MN, Park HH, Lim WK, Shin HJ (2005) Construction and comparison of Escherichia coli whole-cell biosensors capable of detecting aromatic compounds. J Microbiol Meth 60:235–245CrossRefGoogle Scholar
  46. 46.
    Kobatake E, Niimi T, Haruyama T, Ikariyama Y, Aizawa M (1995) Biosensing of benzene derivatives in the environment by luminescent Escherichia coli. Biosens Bioelectron 10:601–605CrossRefGoogle Scholar
  47. 47.
    Paitan Y, Biran I, Shechter N, Biran D, Rishpon J, Ron EZ (2004) Monitoring aromatic hydrocarbons by whole cell electrochemical biosensors. Anal Biochem 335:175–183CrossRefGoogle Scholar
  48. 48.
    Zeinoddini M, Khajeh K, Behzadian F, Hosseinkhani S, Saeedinia AR, Barjesteh H (2010) Design and characterization of an aequorin-based bacterial biosensor for detection of toluene and related compounds. Photochem Photobiol 86:1071–1075CrossRefGoogle Scholar
  49. 49.
    Sanchez-Romero JM, Diaz-Orejas R, de Lorenzo V (1998) Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains. Appl Environ Microbiol 64:4040–4046Google Scholar
  50. 50.
    de Lorenzo V, Herrero M, Sánchez JM, Timmis KN (1998) Mini-transposons in microbial ecology and environmental biotechnology. FEMS Microbiol Ecol 27:211–224Google Scholar
  51. 51.
    Miyamoto-Shinohara Y, Sukenobe J, Imaizumi T, Nakahara T (2008) Survival of freeze-dried bacteria. J Gen Appl Microbiol 54:9–24CrossRefGoogle Scholar
  52. 52.
    Palmfeldt J, Radstrom P, Hahn-Hagerdal B (2003) Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis. Cryobiology 47:21–29CrossRefGoogle Scholar
  53. 53.
    Carvalho AS, Silva J, Ho P, Teixeira P, Malcata FX, Gibbs P (2004) Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus. Biotechnol Prog 20:248–254CrossRefGoogle Scholar
  54. 54.
    Labana S, Pandey G, Paul D, Sharma NK, Basu A, Jain RK (2005) Pot and field studies on bioremediation of p-nitrophenol contaminated soil using Arthrobacter protophormiae rkj100. Environ Sci Technol 39:3330–3337CrossRefGoogle Scholar
  55. 55.
    Baalawy SS (1966) The relative effectiveness of corncobs and polyester granules as vehicles for bayluscide. Bull World Health Organ 35:451Google Scholar
  56. 56.
    Raina V, Suar M, Singh A, Prakash O, Dadhwal M, Gupta SK, Dogra C, Lawlor K, Lal S, van der Meer JR, Holliger C, Lal R (2008) Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum b90a. Biodegradation 19:27–40CrossRefGoogle Scholar
  57. 57.
    Daunert S, Barrett G, Feliciano JS, Shetty RS, Shrestha S, Smith-Spencer W (2000) Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 100:2705–2738CrossRefGoogle Scholar
  58. 58.
    Chinalia FA, Paton GI, Killham KS (2008) Physiological and toxicological characterization of an engineered whole-cell biosensor. Bioresour Technol 99:714–721CrossRefGoogle Scholar
  59. 59.
    Strosnider H (2003) Whole-cell bacterial biosensors and the detection of bioavailable arsenic. Office of Solid Waste and Emergency Response, US EPA, WashingtonGoogle Scholar
  60. 60.
    Paitan Y, Biran D, Biran I, Shechter N, Babai R, Rishpon J, Ron EZ (2003) On-line and in situ biosensors for monitoring environmental pollution. Biotechnol Adv 22:27–33CrossRefGoogle Scholar
  61. 61.
    Matz C, Kjelleberg S (2005) Off the hook -how bacteria survive protozoan grazing. Trends Microbiol 13:302–307CrossRefGoogle Scholar
  62. 62.
    Pedahzur R, Rosen R, Belkin S (2004) Stabilization of recombinant bioluminescent bacteria for biosensor applications. Cell Preserv Technol 2:260–269CrossRefGoogle Scholar
  63. 63.
    Shin HJ, Park HH, Lim WK (2005) Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change. J Biotechnol 119:36–43CrossRefGoogle Scholar
  64. 64.
    Manzanera M, Garcia de Castro A, Tondervik A, Rayner-Brandes M, Strom AR, Tunnacliffe A (2002) Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440. Appl Environ Microbiol 68:4328–4333CrossRefGoogle Scholar
  65. 65.
    Bozoglu TF, Ozilgen M, Bakir U (1987) Survival kinetics of lactic acid starter cultures during and after freeze drying. Enz Microb Technol 9:531–537CrossRefGoogle Scholar
  66. 66.
    Ferrer ML, Yuste L, Rojo F, del Monte F (2003) Biocompatible sol-gel route for encapsulation of living bacteria in organically modified silica matrixes. Chem Mater 15:3614–3618CrossRefGoogle Scholar
  67. 67.
    Gill I, Ballesteros A (2000) Bioencapsulation within synthetic polymers (part 2): non-sol-gel protein-polymer biocomposites. Trends Biotechnol 18:469–479CrossRefGoogle Scholar
  68. 68.
    Nassif N, Bouvet O, Noelle Rager M, Roux C, Coradin T, Livage J (2002) Living bacteria in silica gels. Nat Mater 1:42–44CrossRefGoogle Scholar
  69. 69.
    Rajan Premkumar J, Rosen R, Belkin S, Lev O (2002) Sol-gel luminescence biosensors: encapsulation of recombinant E. coli reporters in thick silicate films. Anal Chim Acta 462:11–23CrossRefGoogle Scholar
  70. 70.
    Amoura M, Nassif N, Roux C, Livage J, Coradin T (2007) Sol-gel encapsulation of cells is not limited to silica: long-term viability of bacteria in alumina matrices. Chem Commun 39:4015–4017CrossRefGoogle Scholar
  71. 71.
    Manohar S, Kim CK, Karegoudar TB (2001) Enhanced degradation of naphthalene by immobilization of Pseudomonas sp. strain ngk1 in polyurethane foam. Appl Microbiol Biotech 55:311–316CrossRefGoogle Scholar
  72. 72.
    Papi RM, Chaitidou SA, Trikka FA, Kyriakidis DA (2005) Encapsulated Escherichia coli in alginate beads capable of secreting a heterologous pectin lyase. Microb Cell Fact 4:35CrossRefGoogle Scholar
  73. 73.
    Ben-Dov E, Kramarsky-Winter E, Kushmaro A (2009) An in situ method for cultivating microorganisms using a double encapsulation technique. FEMS Microbiol Ecol 68:363–371CrossRefGoogle Scholar
  74. 74.
    Ouyang W, Chen H, Jones ML, Metz T, Haque T, Martoni C, Prakash S (2004) Artificial cell microcapsule for oral delivery of live bacterial cells for therapy: design, preparation, and in vitro characterization. J Phar Pharmaceu Scie 7:315–324Google Scholar
  75. 75.
    Cases I, de Lorenzo V (2005) Genetically modified organisms for the environment: stories of success and failure and what we have learned from them. Int Microbiol 8:213–222Google Scholar
  76. 76.
    Moslemy P, Guiot SR, Neufeld RJ (2004) Activated sludge encapsulation in gellan gum microbeads for gasoline biodegradation. Biopro Biosys Eng 26:197–204Google Scholar
  77. 77.
    Moslemy P, Neufeld RJ, Guiot SR (2002) Biodegradation of gasoline by gellan gum-encapsulated bacterial cells. Biotechnol Bioeng 80:175–184CrossRefGoogle Scholar
  78. 78.
    Vilchez S, Tunnacliffe A, Manzanera M (2007) Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress. Extremophiles 12:297–299CrossRefGoogle Scholar
  79. 79.
    Manzanera M, Vilchez S, Tunnacliffe A (2004) Plastic encapsulation of stabilized Escherichia coli and Pseudomonas putida. Appl Environ Microbiol 70:3143–3145CrossRefGoogle Scholar
  80. 80.
    Cole E, Cade D, Benameur H (2008) Challenges and opportunities in the encapsulation of liquid and semi-solid formulations into capsules for oral administration. Adv Drug Deliv Rev 60:747–756CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Systems Biology ProgramCentro Nacional de Biotecnología, CSICMadridSpain

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